Oil-based adjuvants

ABSTRACT

The instant invention provides various formulations comprising combinations of immunostimulating oligonucleotides, polycationic carriers, sterols, saponins, quaternary amines, TLR-3 agonists, glycolipids, and MPL-A or analogs thereof in oil emulsions, use thereof in preparations of immunogenic compositions and vaccines, and use thereof in the treatment of animals.

FIELD OF THE INVENTION

This invention relates generally to novel adjuvant formulations forenhancing the immune response to antigens for use in immunogenic andvaccine compositions. This invention also relates to methods ofpreparation and use of the adjuvant, immunogenic, and vaccinecompositions.

BACKGROUND

Bacterial, viral, and parasitic infections are wide spread in humans andanimals. Diseases caused by these infectious agents are often resistantto antimicrobial pharmaceutical therapy, leaving no effective means oftreatment. Consequently, a vaccinology approach is increasingly used tocontrol infectious disease. A whole infectious pathogen can be madesuitable for use in a vaccine formulation after chemical inactivation orappropriate genetic manipulation. Alternatively, a protein subunit ofthe pathogen can be expressed in a recombinant expression system andpurified for use in a vaccine formulation. Vaccines can be made moreefficacious by including an appropriate adjuvant in the composition.

The term ‘adjuvant’ generally refers to any material that increases thehumoral or cellular immune response to an antigen. Adjuvants are used toaccomplish two objectives: They slow the release of antigens from theinjection site, and they enhance stimulation of the immune system.Traditional vaccines are generally composed of a crude preparation ofinactivated or killed or modified live pathogenic microorganisms. Theimpurities associated with these cultures of pathological microorganismsmay act as an adjuvant to enhance the immune response. However, theimmunity invoked by vaccines that use homogeneous preparations ofpathological microorganisms or purified protein subunits as antigens isoften poor. The addition of certain exogenous materials such as anadjuvant therefore becomes necessary. Further, in some cases, syntheticand subunit vaccines may be expensive to produce. Also, in some cases,the pathogen cannot be grown on a commercial scale, and thus,synthetic/subunit vaccines represent the only viable option. Theaddition of an adjuvant may permit the use of a smaller dose of antigento stimulate a similar immune response, thereby reducing the productioncost of the vaccine. Thus, the effectiveness of some injectablemedicinal agents may be significantly increased when the agent iscombined with an adjuvant.

Many factors must be taken into consideration in the selection of anadjuvant. An adjuvant should cause a relatively slow rate of release andabsorption of the antigen in an efficient manner with minimum toxic,allergenic, irritating, and other undesirable effects to the host. To bedesirable, an adjuvant should be non-viricidal, biodegradable, capableof consistently creating a high level of immunity, capable ofstimulating cross protection, compatible with multiple antigens,efficacious in multiple species, non-toxic, and safe for the host (eg,no injection site reactions). Other desirable characteristics of anadjuvant are that it is capable of micro-dosing, is dose sparing, hasexcellent shelf stability, is amenable to drying, can be made oil-free,can exist as either a solid or a liquid, is isotonic, is easilymanufactured, and is inexpensive to produce. Finally, it is highlydesirable for an adjuvant to be configurable so as to induce either ahumoral or cellular immune response or both, depending on therequirements of the vaccination scenario. However, the number ofadjuvants that can meet the above requirements is limited.

The choice of an adjuvant depends upon the needs for the vaccine,whether it be an increase in the magnitude or function of the antibodyresponse, an increase in cell mediated immune response, an induction ofmucosal immunity, or a reduction in antigen dose. A number of adjuvantshave been proposed, however, none has been shown to be ideally suitedfor all vaccines. The first adjuvant reported in the literature wasFreund's Complete Adjuvant (FCA) which contains a water-in-oil emulsionand extracts of mycobacterium. Unfortunately, FCA is poorly toleratedand it can cause uncontrolled inflammation. Since the discovery of FCAover 80 years ago efforts have been made to reduce the unwanted sideeffects of adjuvants.

Some other materials that have been used as adjuvants include metallicoxides (e.g., aluminum hydroxide), alum, inorganic chelates of salts,gelatins, various paraffin-type oils, synthesized resins, alginates,mucoid and polysaccharide compounds, caseinates, and blood-derivedsubstances such as fibrin clots. While these materials are generallyefficacious at stimulating the immune system, none has been found to beentirely satisfactory due to adverse effects in the host (e.g.,production of sterile abcesses, organ damage, carcinogenicity, orallergenic responses) or undesirable pharmaceutical properties (e.g.,rapid dispersion or poor control of dispersion from the injection site,or swelling of the material).

SUMMARY OF INVENTION

The instant invention provides novel vaccine compositions and adjuvantformulations useful for vaccines.

In the first aspect, the invention provides an adjuvant formulationcomprising an oily phase and an aqueous phase, wherein the oily phasecomprises at least 50% of the formulation v/v, wherein said formulationcomprises at least one of monophosphoryl lipid A (MPL-A) or an analogthereof and an immunostimulatory oligonucleotide, with provisos that a)if said immunostimulatory oligonucleotide is absent, then theformulation comprises a poly I:C, a glycolipid, and, optionally, aquaternary amine; or a polycationic carrier; and b) if saidmonophosphoryl lipid A (MPL-A) or the analog thereof is absent, then theformulation comprises a source of aluminum, and, optionally, apolycationic carrier.

In different embodiments, the oily phase may comprise an oil and,optionally, an oil-soluble emulsifier.

In some embodiments, both said monophosphoryl lipid A (MPL-A) or theanalog thereof are present in the adjuvant formulation. In theseembodiments, the formulation further comprises a sterol (e.g.,cholesterol), a poly I:C, or a combination thereof.

In certain set of embodiments, in addition to the oil and the optionalemulsifier(s), the adjuvant formulations include a combination ofmonophosphoryl lipid A (MPL-A) or an analog thereof, a sterol, and animmunostimulatory oligonucleotide (“TCMO”). The adjuvant formulation mayalso optionally comprise poly I:C (“TCMYO”) and/or a saponin (“QTCMO” or“QTCMYO”, respectively).

In yet further alternative embodiments, in addition to the oil and theoptional emulsifier(s), the adjuvant formulations also include acombination of a quaternary amine, a glycolipid, MPL-A or an analogthereof, and poly I:C (“ODYRM”).

In yet further set of embodiments, in addition to the oil and theoptional emulsifier(s), the adjuvant formulations also include acombination of a saponin, a sterol, a quaternary amine, a polycationiccarrier, with a proviso that if said polycationic carrier is dextranDEAE, then the antigen is not E coli J-5 bacterin (“QCDXO”).

In further embodiments, in addition to the oil and the optionalemulsifier(s), the adjuvant may include the immunostimulatoryoligonucleotide, a source of aluminum, and, optionally, a polycationiccarrier (“TOA” and “TXO-A”, respectively).

In a second aspect, the adjuvant formulation according any of theembodiments recited above, may include an antigen component, thusforming a vaccine composition, with a proviso that the antigen is not EColi J-5 protein if the adjuvant formulation consists of (or consistsessentially of) DEAE dextran, Quil A, Cholesterol, and DDA, or if theadjuvant formulation consists of (or consists essentially of) of DEAEdextran and the immunostimulatory oligonucleotide. In certainembodiments, the vaccines of this aspect contain antigen(s) derived frompathogens affecting cattle, sheep, horses, or swine. In otherembodiments, vaccines of this aspect contain antigen(s) derived frompathogens affecting, poultry or feline animals.

In additional aspects of the invention, different combinations of theantigen compound and the adjuvant formulations are provided.

More specifically, in the third aspect, the invention provides a vaccinecomposition comprising an Eimeria maxima and/or Clostridium perfringensantigen and an adjuvant formulation. In different embodiments of thisthird aspect, the adjuvant formulation may include an oily phase, saidoily phase being present in the amount of at least 50% v/v of thecomposition; a polycationic carrier, and optionally, animmunostimulatory oligonucleotide. In other embodiments of this aspectof the invention, the invention provides a vaccine compositioncomprising an adjuvant component comprising an oily phase, said oilyphase being present in the amount of at least 50% v/v of thecomposition; an immunostimulatory oligonucleotide, a sterol, andmonophosphoryl lipid A (MPL-A) or an analog thereof.

In the fourth aspect, the invention provides a vaccine compositioncomprising a Neospora antigen and an adjuvant formulation. In differentembodiments of the invention according to this aspect, the adjuvantformulation comprises an oily phase, said oily phase being present inthe amount of at least 50% v/v of the composition; and monophosphoryllipid A (MPL-A) or an analog thereof. In other embodiments, the adjuvantformulation comprises an oily phase, said oily phase being present inthe amount of at least 50% v/v of the composition, an immunostimulatoryoligonucleotide and a polycationic carrier.

In the fifth aspect, the invention provides a vaccine compositioncomprising a Chlamydophila abortis antigen and an adjuvant formulationcomprising an oily phase, said oily phase being present in the amount ofat least 50% v/v of the composition; a sterol; an immunostimulatoryoligonucleotide; monophosphoryl lipid A (MPL-A) or an analog thereof;and poly I:C.

In the sixth aspect, the invention provides a vaccine compositioncomprising a Streptococcus uberis (S. uberis) antigen and an adjuvantformulation comprising an oily phase, said oily phase being present inthe amount of at least 50% v/v of the composition; and a polycationiccarrier. In different embodiments of this sixth aspect of the invention,the adjuvant formulation also includes an immunostimulatoryoligonucleotide. Alternatively, or additionally, the adjuvantformulations may include a saponin, a sterol, and a quaternary amine.

In the seventh aspect, the invention provides a vaccine compositioncomprising myostatin as the antigenic component, and an adjuvantformulation, said adjuvant formulation comprising an oily phase, saidoily phase being present in the amount of at least 50% v/v of thecomposition; an immunostimulatory oligonucleotide and either: apolycationic carrier; or MPL-A or an analog thereof. In a set ofembodiments according to this aspect of the invention, the adjuvantformulation comprises MPL-A or the analog thereof. In some embodimentsof this set, the adjuvant formulation contains less than 0.5 ug of asterol per 50 ul of said vaccine composition, and preferably does notcontain cholesterol. The choice of myostatin depends on the subjectspecies. In one selected embodiment, the select species is chicken andthe source of myostatin is chicken myostatin.

In the eighth aspect, the invention provides a vaccine compositioncomprising an A. pyogenes (formerly known as Arcanobacterium pyogenes,Actinomyces pyogenes or Corynebacterium pyogenes; now known asTrueperella pyogenes) antigen and an adjuvant formulation, wherein theadjuvant formulation comprises an oily phase, said oily phase beingpresent in the amount of at least 50% v/v of the composition; animmunostimulatory oligonucleotide and a polycationic carrier.

In the ninth aspect, the invention provides a vaccine compositioncomprising an E coli antigen, a BRV antigen or a BCV antigen, and anadjuvant formulation, wherein said adjuvant formulation comprises anoily phase present in the amount of at least 50% v/v of said vaccinecomposition, an immunostimulatory oligonucleotide and at least one of apolycationic carrier and a source of aluminum.

In the tenth aspect, the invention provides a vaccine compositioncomprising a Rhipicephalus microplus antigen and an adjuvant, saidadjuvant being selected from the group consisting of: a)an aqueousadjuvant comprising an immunostimulatory oligonucleotide, a saponin, asterol, a quaternary amine, a polyacrylic polymer, and a glycolipid; andb)an oil-based adjuvant, comprising an oily phase present in the amountof at least 50% v/v of the vaccine composition and comprising animmunostimulatory oligonucleotide and a polycationic carrier.

In the eleventh aspect, the invention provides a vaccine compositioncomprising a Foot-and-Mouth Disease Virus (FMDV) antigen and an adjuvantformulation, said adjuvant formulation comprising an oily phase presentin the amount of at least 50% v/v of said vaccine composition, animmunostimulatory oligonucleotide and a polycationic carrier. Indifferent embodiments, the Foot-and-Mouth Disease Virus antigen may beof either wild-type FMDV, genetically modified and/or attenuated FMDVstrains, or recombinantly expressed FMDV structural proteins such asvirus like particles (VLPs) of serotypes A, C, O, Asia1, SAT1, SAT2, orSAT3.

In the twelfth aspect, the invention provides a method of generation ofdiagnostic or therapeutic antibodies, the method comprising immunizing asource animal with the adjuvant formulation according to any of theembodiments according to the first aspect of the invention, and antigen,followed by extracting a source of the antibodies from the source animaland if needed, purifying the antibodies.

In certain embodiments, the source animal is a rat, a mouse, a guineapig, a hamster, a cattle animal, a goat, a rabbit, a hourse a swineanimal or an ovine. In some other embodiments, the source animal is acat or a dog.

In some embodiments, particularly suitable for polyclonal antibodies,the source of the antibodies is a serum or milk. In embodiments suitablefor monoclonal antibodies, the suitable source of antibodies is a spleencell.

In certain embodiments, the adjuvant formulation comprises animmunostimulatory oligonucleotide and a polycationic carrier. Theadjuvant may optionally contain a source of aluminum, comprising thesource of aluminum, which may be an aluminum hydroxide gel. In certainembodiments, the immunostimulatory oligonucleotide is a CpG and thepolycationic carrier is DEAE dextran.

In certain embodiments, the antigen may be selected from FeLVgp70,Bovine Parainfluenza-3 BPI-3 (HN protein), Histophilus somni p31,Bordetella FHA, Parapox, BVDV1 gp53, BVDV2 gp53, Clostridia toxins,Canine Circovirus, Brachyspira hyodysenteriae (swine species) Antigens;whole cell inactivated and Pepsin Digest inactivated.

The invention also provides the methods of use of the vaccines accordingto the third through twelfth aspects of the instant invention.

DETAILED DESCRIPTION OF SELECTED EMBODIMENTS

“About” or “approximately,” when used in connection with a measurablenumerical variable, refers to the indicated value of the variable and toall values of the variable that are within the experimental error of theindicated value (e.g., within the 95% confidence interval for the mean)or within 10 percent of the indicated value, whichever is greater,unless about is used in reference to time intervals in weeks where“about 3 weeks,” is 17 to 25 days, and about 2 to about 4 weeks is 10 to40 days.

“Adjuvant” means any substance that increases the humoral or cellularimmune response to an antigen. Adjuvants are generally used toaccomplish two objectives: the controlled release of antigens from theinjection site, and the stimulation of the immune system.

“Adjuvant formulation” refers to formulations having adjuvantingproperties.

“Alkyl” refers to both straight and branched saturated hydrocarbonmoieties.

“Amine” refers to a chemical compound containing nitrogen. Amines are agroup of compounds derived from ammonia by substituting hydrocarbongroups for the hydrogen atoms. “Quaternary amine” refers to an ammoniumbased compound with four hydrocarbon groups.

“Antibody” refers to an immunoglobulin molecule that can bind to aspecific antigen as the result of an immune response to that antigen.Immunoglobulins are serum proteins composed of “light” and “heavy”polypeptide chains having “constant” and “variable” regions and aredivided into classes (e.g., IgA, IgD, IgE, IgG, and IgM) based on thecomposition of the constant regions.

“Antigen” or “immunogen” refers to any substance that is recognized bythe animal's immune system and generates an immune response. The termincludes killed, inactivated, attenuated, or modified live bacteria,viruses, or parasites. The term “antigen” also includes polynucleotides,polypeptides, recombinant proteins, synthetic peptides, protein extract,cells (including tumor cells), tissues, polysaccharides, or lipids, orfragments thereof, individually or in any combination thereof. The termantigen also includes antibodies, such as anti-idiotype antibodies orfragments thereof, and to synthetic peptide mimotopes that can mimic anantigen or antigenic determinant (epitope).

“Bacterin” means a suspension of one or more killed bacteria which maybe used as a component of a vaccine or immunogenic composition.

“Buffer” means a chemical system that prevents change in theconcentration of another chemical substance, e.g., proton donor andacceptor systems serve as buffers preventing marked changes in hydrogenion concentration (pH). A further example of a buffer is a solutioncontaining a mixture of a weak acid and its salt (conjugate base) or aweak base and its salt (conjugate acid).

“Cellular immune response” or “cell mediated immune response” is onemediated by T-lymphocytes or other white blood cells or both, andincludes the production of cytokines, chemokines and similar moleculesproduced by activated T-cells, white blood cells, or both; or a Tlymphocyte or other immune cell response that kills an infected cell.

“Companion animals” refers to dogs, cats and equines.

“Consisting essentially” as applied to the adjuvant formulations refersto formulation which does not contain unrecited additional adjuvantingor immunomodulating agents in the amounts at which said agent exertmeasurable adjuvanting or immunomodulating effects.

“Delayed type hypersensitivity (DTH)” refers to an inflammatory responsethat develops 24 to 72 hours after exposure to an antigen that theimmune system recognizes as foreign. This type of immune responseinvolves mainly T cells rather than antibodies (which are made by Bcells).

“Dose” refers to a vaccine or immunogenic composition given to asubject. A “first dose” or “priming vaccine” refers to the dose of sucha composition given on Day 0. A “second dose” or a “third dose” or an“annual dose” refers to an amount of such composition given subsequentto the first dose, which may or may not be the same vaccine orimmunogenic composition as the first dose.

The term “emulsifier” is used broadly in the instant disclosure. Itincludes substances generally accepted as emulsifiers, e.g., differentproducts of TWEEN® or SPAN® product lines (fatty acid esters ofpolyethoxylated sorbitol and fatty-acid-substituted sorbitansurfactants, respectively), and different solubility enhancers such asPEG-40 Castor Oil or another PEGylated hydrogenated oil.

“Humoral immune response” refers to one that is mediated by antibodies.

“Immune response” in a subject refers to the development of a humoralimmune response, a cellular immune response, or a humoral and a cellularimmune response to an antigen. Immune responses can usually bedetermined using standard immunoassays and neutralization assays, whichare known in the art.

“Immunologically protective amount” or “immunologically effectiveamount” or “effective amount to produce an immune response” of anantigen is an amount effective to induce an immunogenic response in therecipient. The immunogenic response may be sufficient for diagnosticpurposes or other testing, or may be adequate to prevent signs orsymptoms of disease, including adverse health effects or complicationsthereof, caused by infection with a disease agent. Either humoralimmunity or cell-mediated immunity or both may be induced. Theimmunogenic response of an animal to an immunogenic composition may beevaluated, e.g., indirectly through measurement of antibody titers,lymphocyte proliferation assays, or directly through monitoring signsand symptoms after challenge with wild type strain, whereas theprotective immunity conferred by a vaccine can be evaluated bymeasuring, e.g., reduction in clinical signs such as mortality,morbidity, temperature number, overall physical condition, and overallhealth and performance of the subject. The immune response may comprise,without limitation, induction of cellular and/or humoral immunity.

“Immunogenic” means evoking an immune or antigenic response. Thus animmunogenic composition would be any composition that induces an immuneresponse.

“Immunostimulatory molecule” refers to a molecule that stimulates anon-antigen-specific immune response.

“Lipids” refers to any of a group of organic compounds, including thefats, oils, waxes, sterols, and triglycerides that are insoluble inwater but soluble in nonpolar organic solvents, are oily to the touch,and together with carbohydrates and proteins constitute the principalstructural material of living cells.

“Pharmaceutically acceptable” refers to substances, which are within thescope of sound medical judgment, suitable for use in contact with thetissues of subjects without undue toxicity, irritation, allergicresponse, and the like, commensurate with a reasonable benefit-to-riskratio, and effective for their intended use.

The term “Poly I:C” refers to naturally occurring polymers ofpolyinosinic:polycytadylic acids as well as synthetic forms thereof,e.g., with stabilized backbone and preferably having TLR-3 agonistactivity.

“Reactogenicity” refers to the side effects elicited in a subject inresponse to the administration of an adjuvant, an immunogenic, or avaccine composition. It can occur at the site of administration, and isusually assessed in terms of the development of a number of symptoms.These symptoms can include inflammation, redness, and abscess. It isalso assessed in terms of occurrence, duration, and severity. A “low”reaction would, for example, involve swelling that is only detectable bypalpitation and not by the eye, or would be of short duration. A moresevere reaction would be, for example, one that is visible to the eye oris of longer duration.

“Room Temperature” means a temperature from 18 to 25° C.

“Saponin” refers to a group of surface-active glycosides of plant origincomposed of a hydrophilic region (usually several sugar chains) inassociation with a hydrophobic region of either steroid or triterpenoidstructure.

“Steroids” refers to any of a group of organic compounds belonging tobiochemical class of lipids, which are easily soluble in organicsolvents and slightly soluble in water. Steroids comprise a four-fusedring system of three fused cyclohexane (six-carbon) rings plus a fourthcyclopentane (five-carbon) ring.

“Sterols” refers to compounds in animals which are biologically producedfrom terpenoid precursors. They comprise a steroid ring structure,having a hydroxyl (OH) group, usually attached to carbon-3. Thehydrocarbon chain of the fatty-acid substituent varies in length,usually from 16 to 20 carbon atoms, and can be saturated or unsaturated.Sterols commonly contain one or more double bonds in the ring structureand also a variety of substituents attached to the rings. Sterols andtheir fatty-acid esters are essentially water insoluble.

“Subject” refers to any animal for which the administration of anadjuvant composition is desired. It includes mammals and non-mammals,including primates, livestock, companion animals, laboratory testanimals, captive wild animals, ayes (including in ova), reptiles, andfish. Thus, this term includes but is not limited to monkeys, humans,swine; cattle, sheep, goats, equines, mice, rats, guinea pigs, hamsters,rabbits, felines, canines, chickens, turkeys, ducks, other poultry,frogs, and lizards.

“TCID₅₀” refers to “tissue culture infective dose” and is defined asthat dilution of a virus required to infect 50% of a given batch ofinoculated cell cultures. Various methods may be used to calculateTCID₅₀, including the Spearman-Karber method which is utilizedthroughout this specification. For a description of the Spearman-Karbermethod, see B. W. Mahy & H. O. Kangro, Virology Methods Manual, p. 25-46(1996).

“Therapeutically effective amount” refers to an amount of an antigen orvaccine that would induce an immune response in a subject receiving theantigen or vaccine which is adequate to prevent or reduce signs orsymptoms of disease, including adverse health effects or complicationsthereof, caused by infection with a pathogen, such as a virus or abacterium. Humoral immunity or cell-mediated immunity or both humoraland cell-mediated immunity may be induced. The immunogenic response ofan animal to a vaccine may be evaluated, e.g., indirectly throughmeasurement of antibody titers, lymphocyte proliferation assays, ordirectly through monitoring signs and symptoms after challenge with wildtype strain. The protective immunity conferred by a vaccine can beevaluated by measuring, e.g., reduction in clinical signs such asmortality, morbidity, temperature number, overall physical condition,and overall health and performance of the subject. The amount of avaccine that is therapeutically effective may vary depending on theparticular adjuvant used, the particular antigen used, or the conditionof the subject, and can be determined by one skilled in the art.

“Treating” refers to preventing a disorder, condition, or disease towhich such term applies, or to preventing or reducing one or moresymptoms of such disorder, condition, or disease.

“Treatment” refers to the act of “treating” as defined above.

“Triterpeniods” refers to a large and diverse class of naturallyoccurring organic molecules, derived from six five-carbon isoprene(2-methyl-1,3-butadiene) units, which can be assembled and modified inthousands of ways. Most are multicyclic structures which differ from oneanother in functional groups and in their basic carbon skeletons. Thesemolecules can be found in all classes of living things.

“Vaccine” refers to a composition that includes an antigen, as definedherein. Administration of the vaccine to a subject results in an immuneresponse, generally against one or more specific diseases. The amount ofa vaccine that is therapeutically effective may vary depending on theparticular antigen used, or the condition of the subject, and can bedetermined by one skilled in the art.

Adjuvant Formulations and Methods of Making

The instant application discloses several adjuvant formulations suitablefor the instant invention. The common feature of these adjuvants is thepresence of oil and one or more emulsifiers, wherein the oily phasecomprises more than 50% of the vaccine composition encompassing theadjuvant formulations disclosed therein.

Multiple oils and combinations thereof are suitable for use of theinstant invention. These oils include, without limitations, animal oils,vegetable oils, as well as non-metabolizable oils. Non-limiting examplesof vegetable oils suitable in the instant invention are corn oil, peanutoil, soybean oil, coconut oil, and olive oil. Non-limiting example ofanimal oils is squalane. Suitable non-limiting examples ofnon-metabolizable oils include light mineral oil, straight chained orbranched saturated oils, and the like.

In a set of embodiments, the oil used in the adjuvant formulations ofthe instant invention is a light mineral oil. As used herein, the term“mineral oil” refers to a mixture of liquid hydrocarbons obtained frompetrolatum via a distillation technique. The term is synonymous with“liquefied paraffin”, “liquid petrolatum” and “white mineral oil.” Theterm is also intended to include “light mineral oil,” i.e., oil which issimilarly obtained by distillation of petrolatum, but which has aslightly lower specific gravity than white mineral oil. See, e.g.,Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pa.: MackPublishing Company, 1990, at pages 788 and 1323). Mineral oil can beobtained from various commercial sources, for example, J. T. Baker(Phillipsburg, Pa.), USB Corporation (Cleveland, Ohio). Preferredmineral oil is light mineral oil commercially available under the nameDRAKEOL®.

Typically, the oily phase is present in an amount from 50% to 95% byvolume; preferably, in an amount of greater than 50% to 85%; morepreferably, in an amount from greater than 50% to 60%, and morepreferably in the amount of greater than 50-52% v/v of the vaccinecomposition. The oily phase includes oil and emulsifiers (e.g., SPAN®80, TWEEN® 80 etc.), if any such emulsifiers are present. The volume ofthe oily phase is calculated as a sum of volumes of the oil and theemulsifier(s). Thus, for example, if the volume of the oil is 40% andthe volume of the emulsifier(s) is 12% of a composition, then the oilyphase would be present at 52% v/v of the composition. Similarly, if theoil is present in the amount of about 45% and the emulsifier(s) ispresent in the amount of about 6% of a composition, then the oily phaseis present at about 51% v/v of the composition.

It also should be understood that since the adjuvants of the instantinvention form only a part of the vaccines of the instant invention,oily phase is present in an amount from 50% to 95% by volume;preferably, in an amount of greater than 50% to 85%; more preferably, inan amount from 50% to 60%, and more preferably in the amount of 50-52%v/v of each of the adjuvants of the instant invention.

In a subset of embodiments, applicable to all adjuvants/vaccines of theinstant invention, the volume percentage of the oil and the oil-solubleemulsifier together is at least 50%, e.g., 50% to 95% by volume;preferably, in an amount of greater than 50% to 85%; more preferably, inan amount from 50% to 60%, and more preferably in the amount of 50-52%v/v of the vaccine composition. Thus, for example and withoutlimitations, the oil may be present in the amount of 45% and thelipid-soluble emulsifier would be present present in the amount ofgreater than 5% v/v. Thus, the volume percentage of the oil and theoil-soluble emulsifier together would be at least 50%.

In yet another subset, applicable to all vaccines of the invention,volume percentage of the oil is over 40%, e.g., 40% to 90% by volume;40% to 85%; 43% to 60%, 44-50% v/v of the vaccine composition.

Emulsifiers suitable for use in the present emulsions include naturalbiologically compatible emulsifiers and non-natural syntheticsurfactants. Biologically compatible emulsifiers include phospholipidcompounds or a mixture of phospholipids. Preferred phospholipids arephosphatidylcholines (lecithin), such as soy or egg lecithin. Lecithincan be obtained as a mixture of phosphatides and triglycerides bywater-washing crude vegetable oils, and separating and drying theresulting hydrated gums. A refined product can be obtained byfractionating the mixture for acetone insoluble phospholipids andglycolipids remaining after removal of the triglycerides and vegetableoil by acetone washing. Alternatively, lecithin can be obtained fromvarious commercial sources. Other suitable phospholipids includephosphatidylglycerol, phosphatidylinositol, phosphatidylserine,phosphatidic acid, cardiolipin, and phosphatidylethanolamine. Thephospholipids may be isolated from natural sources or conventionallysynthesized.

In additional embodiments, the emulsifiers used herein do not includelecithin, or use lecithin in an amount which is not immunologicallyeffective.

Non-natural, synthetic emulsifiers suitable for use in the adjuvantformulations of the present invention include sorbitan-based non-ionicsurfactants, e.g. fatty-acid-substituted sorbitan surfactants(commercially available under the name SPAN® or ARLACEL®), fatty acidesters of polyethoxylated sorbitol (TWEEN®), polyethylene glycol estersof fatty acids from sources such as castor oil (EMULFOR®);polyethoxylated fatty acid (e.g., stearic acid available under the nameSIMULSOL® M-53), polyethoxylated isooctylphenol/formaldehyde polymer(TYLOXAPOL®), polyoxyethylene fatty alcohol ethers (BRIJ®);polyoxyethylene nonphenyl ethers (TRITON® N), polyoxyethyleneisooctylphenyl ethers (TRITON® X). Preferred synthetic surfactants arethe surfactants available under the name SPAN® and TWEEN®, such asTWEEN®-80 (Polyoxyethylene (20) sorbitan monooleate) and SPAN®-80(sorbitan monooleate).

Generally speaking, the emulsifier(s) may be present in the vaccinecomposition in an amount of 0.01% to 40% by volume, preferably, 0.1% to15%, more preferably 2% to 10%.

Additional ingredients present in the instant adjuvant formulationsinclude cationic carriers, immunostimulatory oligonucleotides,monophospholipid A and analogs thereof (MPL-A),Polyinosinic:polycytidylic acid (poly I:C), saponins, quaternaryammoniums, sterols, glycolipids, a source of aluminum (e.g., REHYDRAGEL®or VAC 20® wet gel) and combinations thereof.

Suitable cationic carriers include, without limitations, dextran,dextran DEAE (and derivatives thereof), PEGs, guar gums, chitosanderivatives, polycellulose derivatives like hydroxyethyl cellulose (HEC)polyethylenimene, poly aminos like polylysine and the like.

Suitable immunostimulatory oligonucleotides include ODN (DNA-based), ORN(RNA-based) oligonucleotides, or chimeric ODN-ORN structures, which mayhave modified backbone including, without limitations, phosphorothioatemodifications, halogenations, alkylation (e.g., ethyl- ormethyl-modifications), and phosphodiester modifications. In someembodiments, poly inosinic-cytidylic acid or derivative thereof (polyI:C) may be used.

CpG oligonucleotides are a recently described class ofpharmacotherapeutic agents that are characterized by the presence of anunmethylated CG dinucleotide in specific base-sequence contexts (CpGmotif). (Hansel T T, Barnes P J (eds): New Drugs for Asthma, Allergy andCOPD. Prog Respir Res. Basel, Karger, 2001, vol 31, pp 229-232, which isincorporated herein by reference). These CpG motifs are not seen ineukaryotic DNA, in which CG dinucleotides are suppressed and, whenpresent, usually methylated, but are present in bacterial DNA to whichthey confer immunostimulatory properties.

In selected embodiments, the adjuvants of the instant invention utilizea so-called P-class immunostimulatory oligonucleotide, more preferably,modified P-class immunostimulatory oligonucleotides, even morepreferably, E-modified P-class oligonucleotides. P-classimmunostimulatory oligonucleotides are CpG oligonucleotidescharacterized by the presence of palindromes, generally 6-20 nucleotideslong. The P-Class oligonucleotides have the ability to spontaneouslyself-assemble into concatamers either in vitro and/or in vivo. Theseoligonucleotides are, in a strict sense, single-stranded, but thepresence of palindromes allows for formation of concatamers or possiblystem-and-loop structures. The overall length of P-classimmunostimulatory oligonucleotides is between 19 and 100 nucleotides,e.g., 19-30 nucleotides, 30-40 nucleotides, 40-50 nucleotides, 50-60nucleotides, 60-70 nucleotides, 70-80 nucleotides, 80-90 nucleotides,90-100 nucleotides.

In one aspect of the invention the immunostimulatory oligonucleotidecontains a 5′ TLR activation domain and at least two palindromicregions, one palindromic region being a 5′ palindromic region of atleast 6 nucleotides in length and connected to a 3′ palindromic regionof at least 8 nucleotides in length either directly or through a spacer.

The P-class immunostimulatory oligonucleotides may be modified accordingto techniques known in the art. For example, J-modification refers toiodo-modified nucleotides. E-modification refers to ethyl-modifiednucleotide(s). Thus, E-modified P-class immunostimulatoryoligonucleotides are P-class immunostimulatory oligonucleotides, whereinat least one nucleotide (preferably 5′ nucleotide) is ethylated.Additional modifications include attachment of 6-nitro-benzimidazol,0-Methylation, modification with proynyl-dU, inosine modification,2-bromovinyl attachment (preferably to uridine).

The P-class immunostimulatory oligonucleotides may also contain amodified internucleotide linkage including, without limitations,phosphodiesther linkages and phosphorothioate linkages. Theoligonucleotides of the instant invention may be synthesized or obtainedfrom commercial sources.

P-Class oligonucleotides and modified P-class oligonucleotides arefurther disclosed in published PCT application no. WO2008/068638,published on Jun. 12, 2008. Suitable non-limiting examples of modifiedP-class immunostiumulatory oligonucleotides are provided below (In SEQID NOs 1-10, “*” refers to a phosphorothioate bond and “_” refers to aphosphodiester bond). In SEQ ID NOs 11-14, all bonds are phosphodiesterbonds.

SEQ ID NO: 1 5′ T*C_G*T*C_G*A*C_G*A*T*C_G* G*C*G*C_G*C*G*C*C*G 3′SEQ ID NO: 2 5′ T*C_G*A*C*G*T*C*G*A*T*C*G* G*C*G*C*G*C*G*C*C*G 3′SEQ ID NO: 3 5′ T*C*G*A*C*G*T*C*G*A*T*C*G* G*C*G*C*G*C*G*C*C*G*T 3′SEQ ID NO: 4 5′ JU*C_G*A*C*G*T*C*G*A*T*C* G*G*C*G*C*G*C*G*C*C*G 3′SEQ ID NO: 5 5′ JU*C_G*A*C*G*T*C*G*A*T*C* G*G*C*G*C*G*C*G*C*C* G*T 3′SEQ ID NO: 6 5′ JU*C*G*A*C*G*T*C*G*A*T*C* G*G*C*G*C*G*C*G*C*C* G*T 3′SEQ ID NO: 7 5′ EU*C_G*A*C*G*T*C*G*A*T*C* G*G*C*G*C*G*C*G*C*C*G 3′SEQ ID NO: 8 5′ JU*C_G*T*C*G*A*C*G*A*T*C* G*G*C*G*G*C*C*G*C*C* G*T 3′SEQ ID NO: 9 5′ JU*C*G*T*C*G*A*C*G*A*T*C* G*G*C*G*G*C*C*G*C*C* G*T 3′SEQ ID NO: 10 5′ T*C_G*T*C_G*A*C_G*A*T*C_ G*G*C*G*C_G*C*G*C*C*G 3′SEQ ID NO: 11 5′-UUGUUGUUGUUGUUGUUGUU-3′ SEQ ID NO: 125′-UUAUUAUUAUUAUUAUUAUU-3′ SEQ ID NO: 13 5′-AAACGCUCAGCCAAAGCAG-3′SEQ ID NO: 14 5′-dTdCdGdTdCdGdTdTdTdTrGr UrUrGrUrGrUdTdTdTdT-3′

The amount of P-class immunostimulatory oligonucleotide for use in theadjuvant compositions depends upon the nature of the P-classimmunostimulatory oligonucleotide used and the intended species.

Suitable analogs of MPL-A include, without limitations can be bacterialderived natural LPS altered or unaltered in structure or synthetic,Glucopyranosyl Lipid Adjuvant (GLA), pertactin, varying substitutions at3-O-position of the reducing sugar, synthetic forms of lipid A analogwith low endotoxicity.

Sterols share a common chemical core, which is a steroid ringstructure[s], having a hydroxyl (OH) group, usually attached tocarbon-3. The hydrocarbon chain of the fatty-acid substituent varies inlength, usually from 16 to 20 carbon atoms, and can be saturated orunsaturated. Sterols commonly contain one or more double bonds in thering structure and also a variety of substituents attached to the rings.Sterols and their fatty-acid esters are essentially water insoluble. Inview of these chemical similarities, it is thus likely that the sterolssharing this chemical core would have similar properties when used inthe vaccine compositions of the instant invention. Sterols are wellknown in the art and can be purchased commercially. For examplecholesterol is disclosed in the Merck Index, 12th Ed., p. 369. Suitablesterols include, without limitations, β-sitosterol, stigmasterol,ergosterol, ergocalciferol, and cholesterol.

Suitable saponins include triterpenoid saponins. These triterpenoids agroup of surface-active glycosides of plant origin and share commonchemical core composed of a hydrophilic region (usually several sugarchains) in association with a hydrophobic region of either steroid ortriterpenoid structure. Because of these similarities, the saponinssharing this chemical core are likely to have similar adjuvantingproperties. Triterpenoids suitable for use in the adjuvant compositionscan come from many sources, either plant derived or syntheticequivalents, including but not limited to, Quillaja saponaria, tomatine,ginseng extracts, mushrooms, and an alkaloid glycoside structurallysimilar to steroidal saponins.

If a saponin is used, the adjuvant compositions generally contain animmunologically active saponin fraction from the bark of Quillajasaponaria. The saponin may be, for example, Quil A or another purifiedor partially purified saponin preparation, which can be obtainedcommercially. Thus, saponin extracts can be used as mixtures or purifiedindividual components such as QS-7, QS-17, QS-18, and QS-21. In oneembodiment the Quil A is at least 85% pure. In other embodiments, theQuil A is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%pure.

Quaternary amine compounds are ammonium based compounds with fourhydrocarbon groups. In practice, hydrocarbon groups are generallylimited to alkyl or aryl groups. In a set of embodiments, the quaternaryamine compounds are composed of four alkyl chains, two of which areC10-C20 alkyls and the remaining two are C1-C4 alkyls. In one set ofembodiments, the quaternary amine is Dimethyldioctadecylammoniumbromide, chloride or pharmaceutically acceptable counterion (DDA).

Suitable glycolipids are generally those which activate the Th2response. The glycolipids include, without limitations, thoseencompassed by Formula I and that are generally described in USPublication 20070196384 (Ramasamy et al).

In the structure of Formula I, R¹ and R² are independently hydrogen, ora saturated alkyl radical having up to 20 carbon atoms; X is —CH₂—, —O—or —NH—; R² is hydrogen, or a saturated or unsaturated alkyl radicalhaving up to 20 carbon atoms; R³, R⁴, and R⁵ are independently hydrogen,—SO₄ ²⁻, —PO₄ ²⁻, —COC₁₋₁₀ alkyl; R⁶ is L-alanyl, L-alpha-aminobutyl,L-arginyl, L-asparginyl, L-aspartyl, L-cysteinyl, L-glutamyl, L-glycyl,L-histidyl, L-hydroxyprolyl, L-isoleucyl, L-leucyl, L-lysyl,L-methionyl, L-ornithinyl, L-phenyalany, L-prolyl, L-seryl, L-threonyl,L-tyrosyl, L-tryptophanyl, and L-valyl or their D-isomers.

In a set of embodiments, the suitable glycolipid isN-(2-Deoxy-2-L-leucylamino-b-D-glucopyranosyl)-N-octadecyldodecanoylamideor an acetate thereof.

Aluminum is a known adjuvant or a component of adjuvant formulations andis commercially available in such forms as Reheis, Inc, Brentagalhydrogel REHYDRAGEL® or VAC 20® wet gel. REHYDRAGEL® is a crystallinealuminum oxyhydroxide, known mineralogically as boehmite. It iseffective in vaccines when there is a need to bind negatively chargedproteins. The content of Al₂O₃ ranges from 2% to 10% depending on grade,and its viscosity is 1000-1300 cP. Generally, it may be described as anadsorbent aluminum hydroxide gel. VAC® 20 wet gel is a white or almostwhite, translucent, viscous colloidal gel. In certain embodiments, thecontent of Al₂O₃ is about 2% w/v.

In other embodiments, the source of aluminum can also be prepared byprecipitated aluminum hydroxide processes.

In certain set of embodiments, in addition to the oil and the optionalone or more emulsifiers, the adjuvant formulations also comprise (orconsist essentially, or consist) a combination of monophosphoryl lipid A(MPL-A) or an analog thereof, a sterol, and an immunostimulatoryoligonucleotide. The adjuvants containing these ingredients are referredto as “TCMO”. The TCMO adjuvant formulation may also optionally includepoly I:C (“TCMYO”) and/or a saponin. Thus, adjuvant formulationscomprising, or consisting essentially of, or consisting of a combinationof monophosphoryl lipid A (MPL-A) or an analog thereof, a sterol, and animmunostimulatory oligonucleotide and saponin are referred to as“QTCMO.” In addition, the adjuvant formulations may also include polyI:C. Such adjuvants are referred to as “QTCMYO”.

In a set of embodiments, TCMO adjuvants comprise light mineral oil inthe amount of 40% to 50% v/v of the total volume of the vaccinecomposition. The emulsifiers include TWEEN-80 and SPAN-80, total amount0.1% to 40% v/v of the total volume of the vaccine composition, providedthat sorbitan monooleate and oil together comprise about 50.5% to 52%v/v of the composition. The immunostimulatory oligonucleotide is an ODN,preferably, a palindrome containing ODN, optionally, with a modifiedbackbone.

In certain embodiments, one dose of TCMO contains between about 1 ug andabout 400 ug of the immunostimulating oligonucleotide, between about 1ug and about 1000 ug of the sterol, between about 0.1 ug and 500 ugMPL-A or the analog thereof.

The amounts of other compounds per dose are selected based on thesubject species.

For example, in some embodiments suitable for cattle, sheep or adultswine, one dose of TCMO would contain between about 50 and 400 ug (e.g.,50-300, or 100-250 ug, or about 50 to about 100 ug for adult pigs andabout 100 to about 250 ug for cattle) of the immunostimulatoryoligonucleotide, between about 100 and about 1000 ug (e.g., 200-1000,250-700 ug, or about 400-500 ug) of the sterol, such as cholesterol, andbetween about 5 and about 500 ug (e.g., 5-100 ug, or 5-50 ug, or 10-25ug) of MPL-A or the analog thereof.

In some embodiments suitable for companion animals or piglets, one doseof TCMO would contain between about 5 and 100 ug (e.g., 10-80, or 20-50ug) of the immunostimulatory oligonucleotide, between about 5 and 100 ug(e.g., 10-80, or 20-50 ug) of the sterol such as cholesterol, andbetween about 0.5 and about 200 ug (e.g., 1-100 ug, or 5-50 ug, or 5-20ug) of MPL-A or the analog thereof.

In some embodiments suitable for poultry, one dose of TCMO adjuvantwould contain between about 0.1 and about 5 ug (e.g., 0.5-3 ug, or0.9-1.1 ug) of immunostimulatory oligonucleotide, between about 0.5 andabout 50 ug (e.g., 1-20 ug, or 1-10 ug) of the sterol such ascholesterol, and between about 0.1 to 10 ug (e.g., 0.5-5 ug, or 1-5 ug)of MPLA or the analog thereof.). MPL-A is present in the amount of 0.1ug/dose to 2,000 ug/dose.

In certain embodiments, TCMO adjuvants are prepared as follows:

-   -   a) Sorbitan monooleate, MPL-A and cholesterol are dissolved in        light mineral oil. The resulting oil solution is sterile        filtered;    -   b) The immunostimulatory oligonucleotide and        Polyoxyethylene (20) sorbitan monooleate are dissolved in        aqueous phase, thus forming the aqueous solution;    -   c) The aqueous solution is added to the oil solution under        continuous homogenization thus forming the adjuvant formulation        TCMO.

In TCMYO adjuvants, the cholesterol, oil, optional emulsifiers, MPL-A,and the immunostimulatory oligonucleotides are present as in the TCMOadjuvant formulation for the respective species. Poly I:C may be presentgenerally in the amount between about 1 ug and about 100 ug per dose.

More specifically, poly I:C may be present in the amount of 5-100 ug perdose (e.g., 5-50 ug, or 10-30 ug) in certain embodiments suitable forcattle, adult swine, or sheep. In certain embodiments suitable forcompanion animals or piglets, one dose of TCMYO contains between about 1and about 50 ug (e.g., 5-50 ug, or 10-20 ug) of poly I:C. In certainembodiments suitable for poultry vaccines, one dose of TCMYO containsbetween about 1 and about 10 ug (e.g., 1-5 ug, or 3-5 ug) of poly I:C.

In certain embodiments, TCMYO adjuvants are prepared similarly to theTCMO adjuvants, and the poly I:C is added to the aqueous solution.

In a set of embodiments, in QTCMO adjuvants, the cholesterol, oil,optional emulsifiers, MPL-A, and the immunostimulatory oligonucleotidesare present as in the TCMO adjuvant formulation for the respectivespecies. A saponin is preferably Quil A or a purified fraction thereof,and may be present in the amounts of between about 0.1 ug and about 1000ug per dose.

More specifically the saponin may be present in the amount of of 0.1 to5 ug per 50 ul of the vaccine composition (e.g., 0.5-30 ug per 50 ul ofthe composition, or more preferably 1-10 ug) per dose in certainembodiments suitable for poultry vaccines. In certain embodimentssuitable for applications in companion animals and piglets, the saponin,e.g., Quil A or a purified fraction thereof is present in the amountsbetween about 10 and about 100 ug per dose (e.g., 10-50 ug or 20-50 ugper dose). In certain embodiments suitable for cattle, adult swine, orsheep, the saponin, such as Quil A or a purified fraction thereof, ispresent in the amount of between about 100 and about 1000 ug per dose(e.g., 200-800 ug, or 250-500 ug per dose).

In certain embodiments, QTCMO adjuvants are prepared similarly to TCMOadjuvants, and the saponin is added to the aqueous solution.

In a set of embodiments, in QTCMYO adjuvants, the saponin is present asin QTCMO adjuvant, and the rest of the ingredients are present as inTCMYO, for the respective species.

In certain embodiments, QTCMYO adjuvants are prepared similarly to TCMYOadjuvants, and the saponin is added to the aqueous solution.

In alternative embodiments, in addition to the oil and the optionalemulsifier(s), the adjuvant formulations also comprise (or consistessentially of, or consist of) a combination of monophosphoryl lipid A(MPL-A) or an analog thereof and a polycationic carrier. These adjuvantsare referred to as “XOM”.

In a set of embodiments, in XOM adjuvants for companion animals orpiglets, the polycationic carrier is present in the amount of 1-50 mgper dose (e.g., 1-25 mg per dose, or 10-25 mg per dose), and the MPL-Aor the analog thereof is present in the amount of between about 1-50 ugper dose (e.g., 1-25 ug per dose, or 10-25 ug per dose).

In certain embodiments suitable for cattle, sheep and adult pigs, thepolycationic carrier is present in the amount of between about 5 andabout 500 mg per dose (e.g., 10-500 mg, or 10-300 mg, or 50-200 mg perdose) and the MPL-A or the analog thereof is present in the amount ofbetween about 1 and about 100 ug per dose (e.g., 5-100 ug, or 5-50 ug,or 10-30 ug).

In certain embodiments suitable for companion animals and piglets, thepolycationic carrier is present in the amount of between about 1 andabout 50 mg per dose (e.g., 1-25 mg per dose, or 10-25 mg per dose), andMPL-A or the analog thereof is present in the amount of between about0.5 and about 200 ug (e.g., 1-100 ug, or 5-50 ug, or 5-20 ug) per dose.

In certain embodiments suitable for poultry vaccines, the polycationiccarrier is present in the amount of between 0.5 and 25 mg per dose(e.g., 1-20 mg, or 1-10 mg or 5-10 mg), and the MPL-A or the analogthereof is present in the amount between about 0.5 and 10 ug per dose(e.g., 1-10 ug, or 1-5 ug, or 2-5 ug).

In certain embodiments, XOM adjuvants are prepared as follows:

-   -   a) Sorbitan monooleate, MPL-A and cholesterol are dissolved in        light mineral oil. The resulting oil solution is sterile        filtered;    -   b) Dextran DEAE and Polyoxyethylene (20) sorbitan monooleate are        dissolved in aqueous phase, thus forming the aqueous solution;    -   c) The aqueous solution is added to the oil solution under        continuous homogenization thus forming the adjuvant formulation        XOM.

In additional alternative embodiments, in addition to the oil and theemulsifier(s), the adjuvant formulations also comprise (or consistessentially of, or consist of) a combination of an immunostimulatoryoligonucleotide and a polycationic carrier, with a proviso that if saidpolycationic carrier is dextran DEAE, then the antigen is not E coli J-5bacterin. These adjuvants are referred to as “TXO”. In certainembodiments, vaccines adjuvanted with TXO contain antigen(s) comprisingpathogens affecting cattle, sheep, horses or swine. In otherembodiments, the antigens are derived from said pathogens. In otherembodiments, vaccines adjuvanted with TXO contain antigen(s) comprisingpathogens affecting, poultry or cats, or the antigens may be derivedfrom such pathogens. In a set of embodiments, the TXO adjuvants may alsoinclude a source of aluminum, such as Al(OH)₃ gel. The TXO adjuvantswith aluminum are referred to as “TXO-A”.

In a set of embodiments, in TXO adjuvants, the immunostimulatoryoligonucleotide, preferably an ODN, preferably containing a palindromicsequence, and optionally with a modified backbone, may be present in theamount of 0.5-400 ug per dose, and the polycationic carrier may bepresent in the amount of 0.5-400 mg per dose. The dosages wary dependingon the subject species.

For example, in certain embodiments suitable for cattle, sheep or adultswine, one dose of TXO would comprise between about 50 and 400 ug (e.g.,50-300, or 100-250 ug, or about 50 to about 100 ug for adult pigs andabout 100 to about 250 ug for cattle) of the immunostimulatoryoligonucleotide, and the polycationic carrier may be present in theamount of between about 5 and about 500 mg per dose (e.g., 10-500 mg, or10-300 mg, or 50-200 mg per dose).

In certain embodiments suitable for companion animals or piglets, onedose of TXO would comprise between about 5 and 100 ug (e.g., 10-80 ug,or 20-50 ug) of the immunostimulatory oligonucleotide, while thepolycationic carrier may be present in the amount of 1-50 mg per dose(e.g., 1-25 mg per dose, or 10-25 mg per dose).

In certain embodiments suitable for poultry, one dose of TXO adjuvantwould between about 0.1 and about 5 ug (e.g., 0.5-3 ug, or 0.9-1.1 ug)of immunostimulatory oligonucleotide, and the polycationic carrier maybe present in the amount of between 0.5 and 25 mg per dose (e.g., 1-20mg, or 1-10 mg or 5-10 mg).

In certain embodiments, TXO adjuvants are prepared as follows:

-   -   a) Sorbitan monooleate is dissolved in light mineral oil. The        resulting oil solution is sterile filtered;    -   b) The immunostimulatory oligonucleotide, Dextran DEAE and        Polyoxyethylene (20) sorbitan monooleate are dissolved in        aqueous phase, thus forming the aqueous solution; and    -   c) The aqueous solution is added to the oil solution under        continuous homogenization thus forming the adjuvant formulation        TXO.

In a set of embodiments, in TXO-A adjuvants, the immunostimulatoryoligonucleotide is present as in the TXO adjuvant, the source ofaluminum is present in the amount of up to 40% v/v (e.g., 35%, 30%, 25%,20%, 15%, 10%, 5%, 1%). In a set of embodiments, the source of aluminumis present at 2%-20% v/v of the vaccine composition, more preferablybetween about 5% and about 17% v/v.

In certain embodiments, TXO-A adjuvants are prepared similarly to TXOadjuvants, and the source of aluminum is added to the aqueous solution.

In additional embodiments, the adjuvants of the instant inventioncontain the oil, optional emulsifier(s), the immunostimulatoryoligonucleotide and the source of aluminum. These compounds are presentin the ranges disclosed for TXO-A adjuvant, except that the polycationiccarrier is absent in TOA. TOA adjuvant is prepared similarly to TXOadjuvant, except the aqueous phase contains the source of aluminumrather than DEAE dextran.

In certain embodiments, in addition to the oil and the emulsifier(s),the adjuvant formulations also comprise (or consist essentially of, orconsist of) a combination of a polycationic carrier and a source ofAluminum. This adjuvant is referred to as AXO. These compounds may bepresent in amounts similar to those present in an adjuvant TXO-A for therespective species, and adjuvant AXO may be prepared similarly to TXO-A,but without addition of the immunostimulating oligonucleotide.

In certain other embodiments, in addition to the oil and theemulsifier(s), the adjuvant formulations also comprise (or consistessentially of, or consist of) a combination of a saponin and sterol.This adjuvant is referred to as QCO. The nature and the amounts of theingredients of QCO are similar to the amounts of the saponin, thesterol, the oil and the emulsifier(s) in adjuvant QTCMO. QCO may beprepared by adding an aqueous solution comprising the saponin the steroland, preferably, the water soluble emulsifier into an oily phase,comprising the oil and, preferably, the oil-soluble emulsifier undercontinuous homogenization.

In yet further alternative embodiments, in addition to the oil and theemulsifier(s), the adjuvant formulations also comprise (or consistessentially of, or consist of) a combination of a quaternary amine, aglycolipid, MPL-A or an analog thereof, and poly I:C. These adjuvantsare referred to as “ODYRM”.

In ODYRM adjuvants, the oil is generally a mixture of phospholipids suchas phosphatidyl cholines. AMPHIGEN® is a suitable example of such oil,and would be present in the amount similar to the amount of oil, asdescribed above.

In a set of embodiments, in ODYRM adjuvants, the quaternary amine, e.g.,DDA, is present in the amount of between about 1 ug and about 200 ug perdose, poly I:C is present in the amount of between about 0.5 ug and 100ug per dose, the glycolipid is present in the amount of between about0.5 ug and about 2000 ug per dose, and the MPL-A or the analog thereofis present in the amount of between about 0.5 ug and 100 ug per dose.

More specifically, in certain embodiments suitable for administration tocattle, adult swine, or sheep, the quaternary amine may be present inthe amount of between about 50 ug and about 200 ug per dose (e.g.,50-150 ug, or about 100 ug), poly I:C may be present in amounts ofbetween about 1 ug and about 100 ug per dose (e.g., 1-50 ug or 5-50 ug),the glycolipid may be present in the amount of between about 500 ug andabout 2000 ug per dose (e.g., 500-100 ug or about 1000 ug), and MPLA orthe analog thereof may be present in the amount of between about 5 ugand about 100 ug per dose (e.g., 5-50 ug, or 10-50 ug).

In certain embodiments suitable for administration to companion animalsand piglets, the quaternary amine may be present in the amount betweenabout 5 and about 500 ug per dose (e.g., 10-100 ug per dose, or 20-50 ugper dose), the poly I:C may be present in the amount of between about 5ug and about 25 ug per dose (e.g., 50-20 ug, or about 10 ug), theglycolipid may be present in the amount of between about 10 and about100 ug per dose (e.g., 20-100 ug or 25-50 ug), and the MPL-A or theanalog thereof may be present in the amount of between about 5 and about50 ug per dose (e.g., 5-20 ug, or 10-20 ug).

In certain other embodiments, suitable for poultry vaccines, one dosewould contain between about 1 ug and about 10 ug of the quaternaryammonium compound (e.g., 5-10 ug, or about 5 ug), between about 0.5 andabout 10 ug of poly I:C (e.g., 1-10 ug or 1-5 ug), between about 0.5 and10 ug of the glycolipid (e.g., 1-10 ug or 5-10 ug or 1-5 ug), andbetween about 0.5 ug and about 5 ug of MPL-A or the analog thereof(e.g., 0.5-5 ug or 1-5 ug).

In certain embodiments, ODYRM adjuvants are prepared as follows:

-   -   a) Sorbitan monooleate, MPL-A are dissolved in light mineral        oil. The resulting oil solution is sterile filtered and        dispersed in water with some surfactant, ethanol and acetic        acid;    -   b) Polyoxyethylene (20) sorbitan monooleate, quaternary amine,        e.g., DDA, and poly I:C are dissolved in aqueous phase, thus        forming the aqueous solution; and    -   c) The aqueous solution is added to the oil solution under        continuous homogenization thus forming the adjuvant formulation        ODYRM.

In yet further set of embodiments, in addition to the oil and theemulsifier(s), the adjuvant formulations also comprise (or consistessentially of, or consist of) a combination of a saponin, a sterol, aquaternary amine, a polycationic carrier, with a proviso that if saidpolycationic carrier is dextran DEAE, then the antigen is not E coli J-5bacterin. These adjuvants are referred to as “QCDXO”.

In QCDXO adjuvants, in certain embodiments, the saponin, e.g., Quil Amay be present in the amounts of between about 0.1 ug and about 1000 ugper dose, the sterol, e.g., cholesterol, is present between about 1 ugand about 1000 ug per dose, the quaternary amine, e.g., DDA, is presentin the amount of between about 1 ug and about 200 ug per dose, and thepolycationic carrier may be present in the amount of 0.5-400 mg perdose. The dosages wary depending on the subject species.

In certain embodiments suitable for cattle, sheep, and adult swine, thesaponin is present in the amount of between about 100 and about 1000 ugper dose (e.g., 200-800 ug, or 250-500 ug per dose), sterol is presentin the amounts between about 100 and about 1000 ug (e.g., 200-1000,250-700 ug, or about 400-500 ug), the quaternary amine may be present inthe amount of between about 50 ug and about 200 ug per dose (e.g.,50-150 ug, or about 100 ug), and the polycationic carrier may be presentin the amount of between about 5 and about 500 mg per dose (e.g., 10-500mg, or 10-300 mg, or 50-200 mg per dose).

In certain embodiments suitable for applications in companion animalsand piglets, the saponin, e.g., Quil A or a purified fraction thereof ispresent in the amounts between about 10 and about 100 ug per dose (e.g.,10-50 ug or 20-50 ug per dose), the sterol is present in the amountsbetween about 5 and 100 ug (e.g., 10-80, or 20-50 ug), the quaternaryamine may be present in the amount between about 5 and about 500 ug perdose (e.g., 10-100 ug per dose, or 20-50 ug per dose), and and thepolycationic carrier may be present in the amount of 1-50 mg per dose(e.g., 1-25 mg per dose, or 10-25 mg per dose.

In some embodiments suitable for poultry vaccines, the saponin may bepresent in the amount of of 0.1 to 5 ug per 50 ul of the vaccinecomposition (e.g., 0.5-30 ug per 50 ul of the composition, or morepreferably 1-10 ug) per dose, the sterol may be present in the amountsbetween about 0.5 and about 50 ug (e.g., 1-20 ug, or 1-10 ug), thequaternary amine may be present in the amount between about 5 and about500 ug per dose (e.g., 10-100 ug per dose, or 20-50 ug per dose) and thepolycationic carrier may be present in the amount of between 0.5 and 25mg per dose (e.g., 1-20 mg, or 1-10 mg or 5-10 mg).

In certain embodiments, QCDXO adjuvants are prepared as follows:

-   -   a) Sorbitan monooleate is dissolved in oil. The resulting oil        solution is sterile filtered;    -   b) Polyoxyethylene (20) sorbitan monooleate, quaternary amine,        e.g., DDA, the polycationic carrier, the sterol and the saponin        are dissolved in aqueous phase, thus forming the aqueous        solution; and    -   c) The aqueous solution is added to the oil solution under        continuous homogenization thus forming the adjuvant formulation        QCDXO.

Sometimes, it is impossible or impracticable to concentrate the antigen,particulary in scaled up commercial applications, and low-concentrationsof antigen solutions have to be used. Thus in some embodiments, thevaccine compositions of the instant invention comprise the adjuvantformulations as described above, wherein the content of the oily phasein these adjuvant formulations is diluted and wherein the vaccinecomposition is a water-in-oil emulsion.

In practice, it is possible to create a water-in-oil emulsion whereinthe oily phase is less than 50% v/v.

Briefly, first, the adjuvant formulation of the instant invention isprepared as described above. In said adjuvant formulaiton, the oilyphase comprises over 50% v/v of the adjuvant formulation. The amounts ofingredients other than the oil and the emulsifier(s) are scaled uprespectively, based on the final target concentration and desireddilution. For example, if one aims to prepare a vaccine compositionwhere the adjuvant formulation comprises 80% v/v, the amounts ofingredients other than the oil are scaled up by the factor of 1.25(I/O.8). The amounts of emulsifiers, if any (e.g., TWEEN® 80 and/orSPAN® 80) do not necessarily need to be scaled up, but preferably, thevolume ratio between the oil and the emulsifier(s) is kept the same inthe adjuvant formulation and in the final vaccine composition.

Antigen solution is then added to the adjuvant formulation.

Water-in-oil emulsion's integrity can be maintained as long as thedispersed spherical water droplets are not present in a moreconcentrated form than the maximum packing fraction for random packingof monodisperse droplets, i.e.: 0.64. See Tadros, Emulsion Formation,Stability and Rheology, 1^(st) ed. 2013, Wiley-VCH GmbH & Co KGaA. Aslong as the total volume fraction occupied by the aqueous droplets doesnot exceed 0.64, i.e.: 64% v/v. Conversely this implies that the oilyphase should not drop below 36% v/v.

Accordingly, in different embodiments of this aspect of the inventionvaccine formulations are provided, comprising the antigen compound, andthe diluted adjuvant formulation according to the previously describedembodiments, wherein the oily phase comprises over 36% of the vaccinecomposition v/v, and wherein the vaccine composition is a water-in-oilemulsion. Without limitations, adjuvant formulations suitable for thisaspect of the invention include TCMO, TCMYO, QTCMO, QTCMYO, XOM, TXO,TXO-A, TAO, AXO, QCO, ODYRM, QCDXO. The volume of the oily phase is, indifferent embodiments, 37% v/v, 38% v/v, 39% v/v, 40% v/v, 41% v/v, 42%v/v, 43% v/v, 44% v/v, 45% v/v, 46% v/v, 47% v/v, 48% v/v, 49% v/v, or50% v/v of the vaccine composition.

The concentration of the oily phase should be sufficiently high tocreate a depot effect and protect the antigen and immunomodulator(s)from a rapid degradation by the host's immune system, preferably 20% ormore v/v of the vaccine composition.

Accordinghly, in another aspect, in the vaccine compositions of theinstant invention, the amounts of the oily phase in the adjuvantformulations are diluted such that the vaccine formulation is anoil-in-water emulsion or a water-in-oil-in-water emulsion, with the oilyphase comprising 20% or more v/v of the vaccine composition. The amountsof ingredients other than the oil and the emulsifiers are scaled uprespectively, based on the final target concentration and desireddilution. For example, to prepare a vaccine composition where theadjuvant formulation comprises 33.3% v/v, the amounts of ingredientsother than the oil and the emulsifier(s) are scaled up by the factor of3 (1/0.333). The amounts of emulsifiers, if any (e.g., TWEEN® 80 and/orSPAN® 80) do not need to be scaled up, but preferably, the volume ratiobetween the oil and the emulsifier(s) is kept the same in the adjuvantformulation and in the final vaccine composition.

In different embodiments, the vaccine composition is an oil-in-wateremulsion or an water-in-oil-in-water emulsion, wherein the oily phasecomprises 21% v/v, 22% v/v, 23% v/v, 24% v/v, 25% v/v, 26% v/v, 27% v/v,28% v/v, 29% v/v, 30% v/v, 31% v/v, 32% v/v, 33% v/v, 34% v/v, 35% v/v,36% v/v, 37% v/v, 38% v/v, 39% v/v, 40% v/v, 41% v/v, 42% v/v, 43% v/v,44% v/v, 45% v/v, 46% v/v, 47% v/v, 48% v/v, 49% v/v, or 50% v/v of thevaccine composition.

Adjuvant formulations suitable for this aspect of the invention includeTCMO, TCMYO, QTCMO, QTCMYO, XOM, TXO, TXO-A, TAO, AXO, QCO, ODYRM,QCDXO, with a proviso that the oily phase in the adjuvant formulationmay be below 50% v/v, but above 20% v/v of the final vaccinecomposition.

Antigens and Diseases

The compositions can contain one or more antigens. The antigen can beany of a wide variety of substances capable of producing a desiredimmune response in a subject, including, without limitations, one ormore of viruses (inactivated, attenuated, and modified live), bacteria,parasites, nucleotides (including, without limitation nucleic-acid basedantigens, e.g., DNA vaccines), polynucleotides, peptides, polypeptides,recombinant proteins, synthetic peptides, protein extract, cells(including tumor cells), tissues, polysaccharides, carbohydrates, fattyacids, teichioc acid, peptidoglycans, lipids, or glycolipids,individually or in any combination thereof.

The antigens used with the adjuvants of the invention also includeimmunogenic fragments of nucleotides, polynucleotides, peptides,polypeptides, that can be isolated from the organisms referred toherein.

Live, modified-live, and attenuated viral strains that do not causedisease in a subject have been isolated in non-virulent form or havebeen attenuated using methods well known in the art, including serialpassage in a suitable cell line or exposure to ultraviolet light or achemical mutagen. Inactivated or killed viral strains are those whichhave been inactivated by methods known to those skilled in the art,including treatment with formalin, betapropriolactone (BPL), binaryethyleneimine (BEI), sterilizing radiation, heat, or other such methods.

Two or more antigens can be combined to produce a polyvalent compositionthat can protect a subject against a wide variety of diseases caused bythe pathogens. Currently, commercial manufacturers of vaccines, as wellas end users, prefer polyvalent vaccine products. While conventionaladjuvants are often limited in the variety of antigens with which theycan be effectively used (either monovalently or polyvalently), theadjuvants described herein can be used effectively with a wide range ofantigens, both monovalently and polyvalently. Thus, the antigensdescribed herein can be combined in a single composition comprising theadjuvants described herein.

Some examples of bacteria which can be used as antigens with theadjuvant compositions include, but are not limited to, Aceinetobactercalcoaceticus, Acetobacter paseruianus, Actinobacillus pleuropneumoniae,Aeromonas hydrophila, Alicyclobacillus acidocaldarius, Arhaeglobusfulgidus, Bacillus pumilus, Bacillus stearothermophillus, Bacillussubtilis, Bacillus thermocatenulatus, Bordetella bronchiseptica,Burkholderia cepacia, Burkholderia glumae, Campylobacter coli,Campylobacter fetus, Campylobacter jejuni, Campylobacterhyointestinalis, Chlamydia psittaci, Chlamydia trachomatis,Chlamydophila spp., Chromobacterium viscosum, Erysipelothrixrhusiopathieae, Listeria monocytogenes, Ehrlichia canis, Escherichiacoli, Haemophilus influenzae, Haemophilus somnus, Helicobacter suis,Lawsonia intracellularis, Legionella pneumophilia, Moraxellsa sp.,Mycobactrium bovis, Mycoplasma hyopneumoniae, Mycoplasma mycoides subsp.mycoides LC, Clostridium perfringens, Odoribacter denticanis,Pasteurella (Mannheimia) haemolytica, Pasteurella multocida,Photorhabdus luminescens, Porphyromonas gulae, Porphyromonas gingivalis,Porphyromonas salivosa, Propionibacterium acnes, Proteus vulgaris,Pseudomonas wisconsinensis, Pseudomonas aeruginosa, Pseudomonasfluorescens C9, Pseudomonas fluorescens SIKW1, Pseudomonas fragi,Pseudomonas luteola, Pseudomonas oleovorans, Pseudomonas sp B11-1,Alcaliges eutrophus, Psychrobacter immobilis, Rickettsia prowazekii,Rickettsia rickettsia, Salmonella enterica all serovars, including forexample: Salmonella enterica Typhimurium, Salmonella enterica Bongori,Salmonella enterica Dublin, Salmonella enterica Choleraseuis, andSalmonella enterica Newport, Serratia marcescens, Spirlina platensis,Staphlyoccocus aureus, Staphyloccoccus epidermidis, Staphylococcushyicus, Streptomyces albus, Streptomyces cinnamoneus, Streptococcusuberis, Streptococcus suis, Streptomyces exfoliates, Streptomycesscabies, Sulfolobus acidocaldarius, Syechocystis sp., Vibrio cholerae,Borrelia burgdorferi, Treponema denticola, Treponema minutum, Treponemaphagedenic, Treponema refringens, Treponema vincentii, Treponemapalladium, Trueperella pyogenes and Leptospira species, such as theknown pathogens Leptospira canicola, Leptospira grippotyposa, Leptospirahardjo, Leptospira borgpetersenii hardjo-bovis, Leptospiraborgpetersenii hardjo-prajitno, Leptospira interrogans, Leptospiraicterohaemorrhagiae, Leptospira pomona, and Leptospira bratislava, andcombinations thereof.

Both inactivated viruses and attenuated live viruses may be used in theadjuvant compositions. Some examples of viruses which can be used asantigens include, but are not limited to, Avian herpesviruses, Bovineherpesviruses, Canine herpesviruses, Equine herpesviruses, Feline viralrhinotracheitis virus, Marek's disease virus, Ovine herpesviruses,Porcine herpesviruses, Porcine Epidemic Diarrhea virus (PEDv),Pseudorabies virus, Avian paramyxoviruses, Bovine respiratory syncytialvirus, Canine distemper virus, Canine parainfluenza virus, canineadenovirus, canine parvovirus, Bovine Parainfluenza virus 3, Ovineparainfluenza 3, Rinderpest virus, Border disease virus, Bovine viraldiarrhea virus (BVDV), BVDV Type I, BVDV Type II, Classical swine fevervirus, Avian Leukosis virus, Bovine immunodeficiency virus, Bovineleukemia virus, Bovine tuberculosis, Equine infectious anemia virus,Feline immunodeficiency virus, Feline leukemia virus (FeLV), NewcastleDisease virus, Ovine progressive pneumonia virus, Ovine pulmonaryadenocarcinoma virus, Canine coronavirus (CCV), pantropic CCV, Caninerespiratory coronavirus, Bovine coronavirus, Feline Calicivirus, Felineenteric coronavirus, Feline infectious peritonitis, virus, Porcineepidemic diarrhea virus, Porcine hemagglutinating encephalomyletitisvirus, Porcine parvovirus, Porcine Circovirus (PCV) Type I, PCV Type II,Porcine Reproductive and Respiratory Syndrome (PRRS) Virus,Transmissible gastroenteritis virus, Turkey coronavirus, Bovineephemeral fever virus, Rabies, Rotovirus, Vesicular stomatitis virus,lentivirus, Avian influenza, Rhinoviruses, Equine influenza virus, Swineinfluenza virus, Canine influenza virus, Feline influenza virus, Humaninfluenza virus, Eastern Equine encephalitis virus (EEE), Venezuelanequine encephalitis virus, West Nile virus, Western equine encephalitisvirus, human immunodeficiency virus, human papilloma virus, varicellazoster virus, hepatitis B virus, rhinovirus, and measles virus, andcombinations thereof.

Examples of peptide antigens include Bordetella bronchiseptica p68,GnRH, IgE peptides, Fel d1, and cancer antigens, and combinationsthereof. Examples of other antigens include nucleotides, carbohydrates,lipids, glycolipids, peptides, fatty acids, lipoteichoic and teichoicacid, and peptidoglycans, and combinations thereof.

Some examples of parasites which can be used as antigens with theadjuvant compositions include, but are not limited to, Anaplasma,Fasciola hepatica (liver fluke), Coccidia, Eimeria spp., Neosporacaninum, Toxoplasma gondii, Giardia, Dirofilaria (heartworms),Ancylostoma (hookworms), Cooperia, Haemonchus contortus (Barber poleworm)Ostertagia ostertagi(stomach worm), Dictyocaulus viviparous (lungworms), Trypanosoma spp., Leishmania spp., Trichomonas spp.,Cryptosporidium parvum, Babesia, Schistosoma, Taenia, Strongyloides,Ascaris, Trichinella, Sarcocystis, Hammondia, and Isopsora, andcombinations thereof. Also contemplated are external parasitesincluding, but not limited to, ticks, including Ixodes, Rhipicephalus,Dermacentor, Amblyomma, Boophilus, Hyalomma, and Haemaphysalis species,and combinations thereof.

The amount of antigen used to induce an immune response will varyconsiderably depending on the antigen used, the subject, and the levelof response desired, and can be determined by one skilled in the art.For vaccines containing modified live viruses or attenuated viruses, atherapeutically effective amount of the antigen generally ranges fromabout 10² Tissue Culture Infective Dose (TCID)₅₀ to about 10¹⁰ TCID₅₀,inclusive. For many such viruses, a therapeutically effective dose isgenerally in the range of about 10² TCID₅₀ to about 10⁸ TCID₅₀,inclusive. In some embodiments, the ranges of therapeutically effectivedoses are about 10³ TCID₅₀ to about 10⁶ TCID₅₀, inclusive. In some otherembodiments, the ranges of therapeutically effective doses are about 10⁴TCID₅₀ to about 10⁵ TCID₅₀, inclusive.

For vaccines containing inactivated viruses, a therapeutically effectiveamount of the antigen is generally at least about 100 relative units perdose, and often in the range from about 1,000 to about 4,500 relativeunits per dose, inclusive. In other embodiments, the therapeuticallyeffective amount of the antigen is in a range from about 250 to about4,000 relative units per dose, inclusive, from about 500 to about 3,000relative units per dose, inclusive, from about 750 to about 2,000relative units per dose, inclusive, or from about 1,000 to about 1,500relative units per dose, inclusive.

A therapeutically effective amount of antigen in vaccines containinginactivated viruses can also be measured in terms of Relative Potency(RP) per mL. A therapeutically effective amount is often in the rangefrom about 0.1 to about 50 RP per mL, inclusive. In other embodiments,the therapeutically effective amount of the antigen is in a range fromabout 0.5 to about 30 RP per mL, inclusive, from about 1 to about 25 RPper mL, inclusive, from about 2 to about 20 RP per mL, inclusive, fromabout 3 to about 15 RP per mL, inclusive, or from about 5 to about 10 RPper mL, inclusive.

The number of cells for a bacterial antigen administered in a vaccineranges from about 1×10⁶ to about 5×10¹⁰ colony forming units (CFU) perdose, inclusive. In other embodiments, the number of cells ranges fromabout 1×10⁷ to 5×10¹⁰ CFU/dose, inclusive, or from about 1×10⁸ to 5×10¹⁰CFU/dose, inclusive. In still other embodiments, the number of cellsranges from about 1×10² to 5×10¹⁰ CFU/dose, inclusive, or from about1×10⁴ to 5×10⁹ CFU/dose, inclusive, or from about 1×10⁵ to 5×10⁹CFU/dose, inclusive, or from about 1×10⁶ to 5×10⁹ CFU/dose, inclusive,or from about 1×10⁶ to 5×10⁸ CFU/dose, inclusive, or from about 1×10⁷ to5×10⁹ CFU/dose, inclusive.

The number of cells for a parasite antigen administered in a vaccineranges from about 1×10² to about 1×10¹⁰ per dose, inclusive. In otherembodiments, the number of cells ranges from about 1×10³ to about 1×10⁹per dose, inclusive, or from about 1×10⁴ to about 1×10⁸ per dose,inclusive, or from about 1×10⁵ to about 1×10⁷ per dose, inclusive, orfrom about 1×10⁶ to about 1×10⁸ per dose, inclusive.

It is well known in the art that with conventional adjuvants, asubstantially greater amount of inactivated viruses than modified liveor attenuated viruses is needed to stimulate a comparable level ofserological response. However, it has been surprisingly found that withthe adjuvant compositions described herein, approximately the sameamounts of inactivated virus and modified live virus stimulate similarlevels of serological response. In addition, smaller amounts of modifiedlive, attenuated, and inactivated virus are needed with the adjuvantsdescribed herein when compared with conventional adjuvants to achievethe same level of serological response. These unexpected findingsdemonstrate conservation of resources and reduction of cost duringpreparation of immunogenic and vaccine compositions. For vaccines withwide utility, the manufacture of millions of doses per year is required,so these savings can be substantial.

Administration of the Compositions

Dose sizes of the compositions typically range from about 1 mL to about5 mL, inclusive, depending on the subject and the antigen. For example,for a canine or feline, a dose of about 1 mL is typically used, while incattle a dose of about 2-5 mL is typically used. However, theseadjuvants also can be formulated in microdoses, wherein doses of about100.mu.L can be used.

The routes of administration for the adjuvant compositions includeparenteral, oral, oronasal, intranasal, intratracheal, topical,subcutaneous, intramuscular, transcutaneous, intradermal,intraperitoneal, intraocular, intravenous administration and in ova. Anysuitable device may be used to administer the compositions, includingsyringes, droppers, needleless injection devices, patches, and the like.The route and device selected for use will depend on the composition ofthe adjuvant, the antigen, and the subject, and such are well known tothe skilled artisan.

Use of the Compositions

One of the requirements for any vaccine adjuvant preparation forcommercial use is to establish the stability of the adjuvant solutionfor long periods of storage. Provided herein are adjuvant formulationsthat are easy to manufacture and stable for at least 18 months. In oneembodiment, the formulations are stable for about 18 months. In anotherembodiment, the formulations are stable for between about 18 to about 24months. In another embodiment the formulations are stable for about 24months. Accelerated testing procedures also indicate that theformulations described herein are stable.

An advantageous feature of the present adjuvant compositions is thatthey can be safely and effectively administered to a wide range ofsubjects. In the art, it is expected that combinations of adjuvants willdemonstrate more reactogenicity than the individual components. However,the compositions described herein show decreased reactogenicity whencompared to compositions in which any one or two of the components areused, while the adjuvant effect is maintained. It has also beensurprisingly found that the adjuvant compositions described hereindemonstrate safety improvements when compared with other adjuvantcompositions.

The adjuvant compositions described herein are useful for inducing adesired immune response in a subject. They are efficacious in multiplespecies. A suitable subject is any animal for which the administrationof an adjuvant composition is desired. It includes mammals andnon-mammals, including primates, livestock, companion animals,laboratory test animals, captive wild animals, ayes (including in ova),reptiles, and fish. Thus, this term includes but is not limited tomonkeys, humans, swine; cattle, sheep, goats, equines, mice, rats,guinea pigs, hamsters, rabbits, felines, canines, chickens, turkeys,ducks, other poultry, frogs, and lizards.

The adjuvants described herein can be used to show serologicaldifferentiation between infected and vaccinated animals. Thus, they canbe used in a marker vaccine in which the antigen in the vaccine elicitsin the vaccinated animals a different antibody pattern from that of thewild-type virus. A marker vaccine is generally used in conjunction witha companion diagnostic test which measures the difference in antibodypatterns and demonstrates which animals have been vaccinated and whichanimals are infected with the wild-type virus. Such technology is usefulin the control and eradication of viruses from a subject population.

The present invention also provides novel vaccine compositions useful inprotecting against infection and disease caused by Nipah virus and/orHendra virus, using antigen provided from Hendra virus G protein (andfragments, dimers, multimers, and modified forms thereof), all of whichare adjuvanted as described herein. In certain embodiments, the adjuvantis selected from the group consisting of TXO, TAO, and TXO-A. Suchvaccines are useful in preventing infection and disease in, for example,horses, dogs, swine and humans. In a most preferred embodiment, bothswine and dogs are protected from both Hendra and Nipah virus.

Recurrent outbreaks of NiV resulting in significant numbers of humanfatalities have recently been problematic, see, for example, Butler,Nature, vol. 429, at page 7 (2000); and Gurley et al., EmergingInfectious Diseases, vol. 13(7), pp. 1031-1037 (2007). Case studies havelinked disease in humans to zoonotic transmission from swine, seeParashar et al., J. Infect. Dis. vol 181, pp. 1755-1759 (2000). Hendravirus has also clearly been linked to deaths in humans, via transmissionfrom horses. There is presently one licensed vaccine for the preventionof infection or disease caused by Hendra virus (Equivac® HeV; Zoetis)approved for use in horses, although no licensed vaccine exists forpreventing Nipah virus infection. There remains a need for Nipah virusor Hendra virus vaccines that can be clinically effective.

Paramyxoviruses such as Hendra virus and Nipah virus possess two majormembrane-anchored glycoproteins in the envelope of the viral particle.One glycoprotein is required for virion attachment to receptors on hostcells and is designated as either hemagglutinin-neuraminidase protein(HN) or hemagglutinin protein (H), and the other is glycoprotein (G),which has neither hemagglutination nor neuraminidase activities. Theattachment glycoproteins are type II membrane proteins, where themolecule's amino (N) terminus is oriented toward the cytoplasm and theprotein's carboxy (C) terminus is extracellular. The other majorglycoprotein is the fusion (F) glycoprotein, which is a trimeric class Ifusogenic envelope glycoprotein containing two heptad repeat (HR)regions and a hydrophobic fusion peptide. Hendra virus and Nipah virusinfect cells through a pH-independent membrane fusion process intoreceptive host cells, through the concerted action of their attachment Gglycoprotein and F glycoprotein following receptor binding.

That Hendra virus G glycoprotein could potentially cross protect againstinfection and disease by Nipah virus is suggested by K. Bossart et al.,Journal of Virology, vol. 79, pp. 6690-6702 (2005), and B. Mungall etal., Journal of Virology, vol. 80, pp. 12293-12302 (2006). However,prior work does not provide vaccine compositions that are actuallyclinically effective in this regard, for any mammalian species.Accordingly, the present invention encompasses an immunogeniccomposition comprising Hendra virus G protein, an adjuvant as describedaccording to the practice of the present invention, and one or moreexcipients, in an amount effective to elicit clinically effectiveprotection against Hendra and/or Nipah virus.

In regard of Hendra virus G glycoprotein polypeptides that are useful inthe practice of the present invention, and the recombinant expressionthereof, reference is made to the entire disclosure of publishedinternational patent applications WO 2012/158643 and WO2006/085979 wheresuch information is clearly set forth. Preferred examples of specificHendra virus G protein polypeptides useful herein are disclosed in WO2012/158643, and include, for example: full length G protein (SEQ IDNO:2 thereof); a soluble fragment thereof (encoding amino acids 73-604of SEQ ID NO:2 of WO 2012/158643); and an additional fragment disclosedtherein having an Ig(kappa) leader sequence (SEQ ID NO 16 of WO2012/158643). Generally, the soluble forms of the Hendra virus Gglycoprotein comprises all or part of the ectodomain, and are producedby deleting all or part of the transmembrane domain of the Gglycoprotein, and all or part of the cytoplasmic tail. Preferably, theencoding gene sequence is codon optimized for expression.

In some embodiments, the Hendra G glycoprotein may be in dimeric and/ortetrameric form. Such dimers depend upon the formation of disulfidebonds formed between cysteine residues in the G glycoprotein. Suchdisulfide bonds can correspond to those formed in the native Gglycoprotein, or different disulfide bonds can be formed resulting indifferent dimeric and/or tetrameric forms of the G glycoprotein whichenhance antigenicity. Additionally, non-dimerized and tetramerized formsare also useful according to the practice of the present invention,again taking into account that G glycoprotein provides numerousconformation-dependent epitopes (i.e. that arise from a tertiary threedimensional structure) and that preservation of numerous of such naturalepitopes is accordingly highly preferred so as to impart a neutralizingantibody response.

Generally speaking, construction of expression vectors for the Hendra Gproteins can be as described in Example 1 of WO 2012/158643, withresultant protein expression from CHO cells being as described inExample 2 thereof, or alternatively, using a Vaccinia system (seeExample 3 thereof) or 293 cells (see Example 4 thereof). In a specificpreferred example, the soluble G protein is provided as amino acids73-604 of the native Hendra virus G glycoprotein (see SEQ ID NO: 2 in WO2012/158643). Dimerization thereof occurs spontaneously, concomitantwith expression from CHO cells, and resultant G protein is approximately50% dimer and 50% tetramer, with little remaining monomer. Expression in293F cells leads to about 70% dimer. The resultant protein fractions aremixed with adjuvants as described throughout the present specification.As described in WO2012/158643, preferred doses of antigen for largeanimals are in the range of 50-200 micrograms per dose, with 100micrograms being a most preferred dose. For smaller animals, such asdogs, lesser amounts are needed, such as 25-50 micrograms, as will beappreciated by those skilled in the art.

In addition, adjuvants according to any of the embodiments describedabove may be used for generation of diagnostic or therapeuticantibodies. In this aspect of the invention, a source animal isimmunized with a formulation containing the adjuvant compositions of theinstant invention and an antigen. The choice of the antigen isdetermined by the person who needs to obtain said therapeutic ordiagnostic antibodies and includes, without limitations, viruses,bacteria, viral particles, extracts, recombinant antigens, cell wallstructures and the like. Antigens may also include venoms forpreparation of medicines against snake bites.

The antigens suitable for this aspect of the invention may be of afeline, a canine, an equine, a porcine, a bovine, an ovine or avianorigin. In certain embodiments, the antigen may be selected fromFeLVgp70, Bovine Parainfluenza-3 BPI-3 (HN protein), Histophilus somnip31, Bordetella FHA, Parapox, BVDV1 gp53, BVDV2 gp53, Clostridia toxins,Canine Circovirus, Brachyspira hyodysenteriae (swine species) Antigens;whole cell inactivated and Pepsin Digest inactivated.

A certain time after immunization, a source of antibodies is extractedfrom the source animal (e.g., mice, rats, hamsters, swine, guinea pigs,rabbits, goats, sheep, poultry, cattle, horses). In certain otherembodiments, the source animal is a cat or a dog. The source ofantibodies ultimately depends on whether monoclonal or polyclonalantibodies are needed. For polyclonal antibodies, one may consider usingserum or milk. For monoclonal antibodies, spleen cells are suitablesource. Such antibodies may be used for diagnostic, research, ortherapeutic purposes, including, without limitations, anti-venom,transplant rejection medications, Serum Neutralization assays, ELISAs,ELISPOTs, Western blots, Cell-based assays, potency assays, andImmunohistochemistry. The invention provides monoclonal and polyclonalantibodies extracted from the source animal for use in diagnostic andtherapeutic applications, including without limitatins, anti-venom,transplant rejection medications, Serum Neutralization assays, ELISAs,ELISPOTs, Western blots, Cell-based assays, potency assays, andlmmunohistochemistry.

In certain embodiments, immunizations with the compositions of theinstant invention would elicit sufficiently high serology titers to thedesired antigen (over 1000, or more preferably, over 5000, or morepreferably, over 10000, or more preferably, over 50000, or morepreferably, over 100000, or more preferably, over 250000, or morepreferably, over 500000, or more preferably, over 1000000) in at leastone animal (preferably at least 2 animals, or at least three animals, orin 50% of treated animals, or in at least 75% of treated animals, or,most preferably, in every animal treated) thus resulting in sufficientamount of antibodies for diagnostic or research applications.

Typically, antibodies of the lmmunoglobulin G (IgG) isotype are used inthese applications, although antibodies of other isotypes, e.g.,lmmunoglobulin M (IgM), are also employed. The antibody sourceultimately depends on whether a polyclonal or a monoclonal antibody isdesired. For polyclonal antibodies, one may use serum or milk as thesource of the antibodies. For monoclonal antibodies, splenocytes is theproper antibody source. Further purification of the antibodies, ifneeded, or preparation of monoclonal antibodies have been described inliterature extensively and one of ordinary skill in the art would haveno undue difficulties in performing these procedures. Further,antibodies may be adapted to the target species, if needed (e.g.,canonized or felinized). Again, the techniques for doing so are wellknown in the art and do not need to be described in this application.

Applications of antibodies

The antibodies would be suitable as reagents for Serum Neutralizationassays, ELISAs, ELISPOTs, Western blots, Cell-based assays, potencyassays, and Immunohistochemistry. These techniques have been known inthe field.

The antibodies of the instant invention may also be used as therapeuticagents, e.g., in transplant rejection, e.g., for generation ofantithymocyte globulin (ATG) agents. Currently, two such agents are onthe market: Atgam® and Thymoglobulin®. Methods of making anti-thymocyteglobulins in general have been described in US20040023340.

It may also be used for the preparation of anti-venom medicines. Inthese embodiments, snake venom components would be used as antigens. Thevenoms and components thereof are also well known in the art.

Animals of many species may be used as source animals, including,without limitations, poultry, mice, rats, hamsters, guinea pigs,rabbits, dogs, cats, sheep, goats, swine, cattle, and equine species.The choice of the source animal depends on the task at hand and judgmentof the person of ordinary skill in the art.

Specific non-limiting embodiments are as follows:

In a first embodiment, the invention provides an adjuvant formulationcomprising an oily phase and an aqueous phase, wherein the oily phasecomprises at least 50% of the formulation v/v, wherein said formulationcomprises at least one of monophosphoryl lipid A (MPL-A) or an analogthereof and an immunostimulatory oligonucleotide, with provisos that:

-   -   a) if said immunostimulatory oligonucleotide is absent, then the        formulation comprises:        -   i. a poly I:C, a glycolipid, and, optionally, a quaternary            amine; or        -   ii. a polycationic carrier;    -   b) if said monophosphoryl lipid A (MPL-A) or the analog thereof        is absent, then the formulation comprises a source of aluminum.

In the second embodiment, the invention provides the adjuvantformulation of the first embodiment, wherein the immunostimulatoryoligonucleotide, if present, is a CpG or an oligoribonucleotide; thepolycationic carrier, if present, is selected from the group consistingof dextran, dextran DEAE (and derivatives thereof), PEGs, guar gums,chitosan derivatives, polycellulose derivatives like hydroxyethylcellulose (HEC) polyethylenimene, poly aminos; and the quaternary amine,if present, is selected from the group consisting of DDA and avridine.

In the third embodiment, the invention provides the adjuvant formulationaccording to the first or the second embodiment, wherein theimmunostimulatory oligonucleotide if present, is the CpG, thepolycationic carrier, if present, is dextran DEAE, and the quaternaryamine, if present, is DDA.

In the fourth embodiment, the inv ention provides the adjuvantformulation according to any one of first through third embodiments,wherein the glycolipid, if present, comprises a compound of formula I

wherein, R¹ and R² are independently hydrogen, or a saturated alkylradical having up to 20 carbon atoms; X is —CH₂—, —O— or —NH—; R² ishydrogen, or a saturated or unsaturated alkyl radical having up to 20carbon atoms; R³, R⁴, and R⁵ are independently hydrogen, —SO₄ ²⁻, —PO₄²⁻, —COC₁₋₁₀ alkyl; R⁶ is L-alanyl, L-alpha-aminobutyl, L-arginyl,L-asparginyl, L-aspartyl, L-cysteinyl, L-glutamyl, L-glycyl, L-histidyl,L-hydroxyprolyl, L-isoleucyl, L-leucyl, L-lysyl, L-methionyl,L-ornithinyl, L-phenyalany, L-prolyl, L-seryl, L-threonyl, L-tyrosyl,L-tryptophanyl, and L-valyl or their D-isomers.

In the fifth embodiment, the invention provides the adjuvant formulationof the fourth embodiment, wherein the glycolipid isN-(2-Deoxy-2-L-leucylamino-b-D-glucopyranosyl)-N-octadecyldodecanoylamideor a salt thereof.

In the sixth embodiment, the invention provides the adjuvant formulationof the fifth embodiment, wherein the salt is an acetate.

In the seventh embodiment, the invention provides the adjuvantformulation of any one of fists thought fourth embodiments, comprisingboth said monophosphoryl lipid A (MPL-A) or the analog thereof, andfurther comprising at least one of a sterol and a poly I:C.

In the eighth embodiment, the invention provides the adjuvantformulation according to the seventh embodiment, comprising the steroland further comprising a saponin.

In the ninth embodiment, the invention provides the adjuvant formulationof any one of the senventh and the eighth embodiments, wherein thesaponin, if present, is a triterpenoid saponin, and the sterol, ifpresent, is selected from the group consisting of ergosterol, lanosteroland cholesterol.

In the tenth embodiment, the invention provides the adjuvant formulationaccording to the ninth embodiment, wherein the saponin, if present, isQuil A, and the sterol, if present, is cholesterol.

In the eleventh embodiment, the invention provides the adjuvantformulation according to the seventh embodiment, comprising the polyI:C, and further comprising at least one of the quaternary amine and theglycolipid.

In the twelfth embodiment, the invention provides the adjuvantformulation of any one of the first-eleventh embodiments, comprising theMPL-A or the analog thereof in the amount of 0.5-100 ug per dose.

In the thirteenth embodiment, the invention provides the adjuvantformulation according to the twelfth embodiment, wherein the MPL-A orthe analog thereof is present in the amount of 5-50 ug per dose, or 5-20ug per dose, or 1-5 ug per dose.

In the fourteenth embodiment, the invention provides the adjuvantformulation of any one of the first-thirteenth embodiments, comprisingthe immunostimulatory oligonucleotide in the amount of 0.5 to 400 ug perdose.

In the fifteenth embodiment, the invention provides the adjuvantformulation of the fourteenth embodiment, wherein the immunostimulatoryoligonucleotide is present in the amount of about 100 to about 250 ugper dose or about 20 to about 50 ug per dose, or about 1 ug per dose.

In the sixteenth embodiment, the invention provides the adjuvantformulation of any one of first through fifteenth embodiments,comprising the polycationic carrier in the amount of between about 0.5and about 400 mg per dose.

In the seventeenth embodiment, the invention provides the adjuvantformulation of the sixteenth embodiment, wherein said polycationiccarrier is present in the amount of 50-300 mg per dose or 1-25 mg perdose, or 1-10 mg per dose.

In the eighteenth embodiment, the invention provides the adjuvantformulation of any one of the first-seventeenth embodiment, comprisingthe glycolipid in the amount of between about 0.5 and about 2000 ug perdose.

In the nineteenth embodiment, the invention provides the adjuvantformulation of the eighteenth embodiment, wherein the glycolipid ispresent in the amount of about 1000 ug per dose, or 25-50 ug per dose,or 1-10 ug per dose.

In the twentieth embodiment, the invention provides the adjuvantformulation of any one of the first-nineteenth embodiments, comprisingthe sterol in the amount of between about 0.1 and about 1000 ug perdose.

In the twenty-first embodiment, the invention provides the adjuvantformulation according to the twentieth embodiment, wherein the sterol ispresent in the amount of 250-500 ug per dose, or 20-50 ug per dose, or1-10 ug per dose.

In the twenty-second embodiment, the invention provides the adjuvantformulation of any one of first through twenty-first embodiment,comprising the saponin in the amount of between 0.1 and 1000 ug perdose.

In the twenty-third embodiment, the invention provides the adjuvantformulation of the twenty-second embodiment, wherein the saponin ispresent in the amount of 250-500 ug per dose, or 20-50 ug per dose, or1-10 ug per dose.

In the twenty-fourth embodiment, the invention provides the adjuvantformulation of any one of first through twenty-third embodiment,comprising the poly I:C is in the amount of between about 0.5 and about100 ug per dose.

In the twenty fifth embodiment, the invention provides the adjuvantformulation of the twenty-fourth embodiment, wherein the poly I:C ispresent in the amount of 5-50 ug per dose, or 5-20 ug per dose, or 1-5ug per dose.

In the twenty-sixth embodiment, the invention provides the adjuvantformulation of any one of first through twenty-fifth embodiment,comprising the source of aluminum, which is an aluminum hydroxide gel.

In the twenty-seventh embodiment, the invention provides the adjuvantformulation of twenty-sixth embodiment, wherein said source of aluminumis present in the amount of 5%-20% v/v of the formulation.

In the twenty-eighth embodiment, the invention provides the adjuvantformulation of the twenty-seventh embodiment, wherein said source ofaluminum is present in the amount of 10% v/v of the formulation.

In the twenty-ninth embodiment, the invention provides the adjuvantformulation of any one of the first through twenty-eighth embodiment,wherein the oily phase comprises an oil and an oil-soluble emulsifier.

In the thirtieth embodiment, the invention provides the adjuvantformulation of any one of the first through the twenty-ninth embodiment,wherein said oily phase is present in the amount of up to 85% v/v.

In the thirty-first embodiment, the invention provides the adjuvantformulation according to the thirtieth embodiment, wherein said oilyphase is present in the amount of 51%.

In the thirty-second embodiment, the invention provides the adjuvantformulation of any one of the twenty-ninth through the thirty-firstembodiments, wherein the oil comprises 40-84% v/v of the formulation,and the oil-soluble emulsifier comprises 1-11% v/v of the formulation.

In the thirty-third embodiment, the invention provides the adjuvantformulation of the thirty-second embodiment, wherein the oil comprises45% v/v of the formulation, and the oil-soluble emulsifier comprises 6%v/v of the formulation.

In the thirty-fourth embodiment, the invention provides the adjuvantformulation according to any one of the first through thirty-thirdembodiment, wherein said oil is selected from the group consisting ofsqualane, vegetable oils, triglycerides, non-metabolizablestraight-chain alkane oils, and any combination thereof.

In the thirty-fifth embodiment, the invention provides the adjuvantformulation according to the thirty-fourth embodiment, wherein said oilis a light mineral oil.

In the thirty-sixth embodiment, the invention provides a vaccinecomposition comprising an effective amount of an antigen and theadjuvant formulation according to any one of the first through thethirty-fifth embodiment, wherein the oily phase of the composition is atleast 50% v/v.

In the thirty-seventh embodiment, the invention provides a vaccinecomposition comprising an effective amount of an antigen and an adjuvantformulation comprising an oily phase and an aqueous phase, wherein theoily phase comprises at least 50% of the formulation v/v, a polycationiccarrier, and

-   -   a. a combination of a saponin and a sterol, and optionally, a        quaternary amine; with provisos that if said adjuvant        formulation consists essentially of DEAE dextran, Quil A,        Cholesterol, and DDA, the antigen is not E coli J-5 bacterin; or    -   b. an immunostimulatory oligonucleotide, with a proviso that if        said adjuvant formulation consists essentially of DEAE dextran        and the immunostimulatory oligonucleotide, the antigen comprises        a pathogen affecting cattle, sheep, horses, or swine or is        derived from said pathogen, and is not E coli J-5 bacterin.

In the thirty-eighth embodiment, the invention provides he vaccinecomposition according to the thirty-seventh embodiment, wherein thesaponin, if present, is a triterpenoid saponin, the sterol, if present,is selected from the group consisting of ergosterol, lanosterol andcholesterol, the polycationic carrier, if present, is selected from thegroup consisting of dextran, dextran DEAE (and derivatives thereof),PEGs, guar gums, chitosan derivatives, polycellulose derivatives likehydroxyethyl cellulose (HEC) polyethylenimene, poly aminos, and thequaternary amine, if present, is selected from the group consisting ofDDA and avridcine.

In the thirty-ninth embodiment, the invention provides the vaccinecomposition according to the thirty-eighth embodiment, wherein thesaponin is Quil A, the sterol is cholesterol, the polycationic carrieris dextran DEAE, and the quaternary amine is DDA.

In the fourtieth embodiment, the invention provides the vaccinecomposition of any one of thirty-seventh though thirty-ninthembodiments, wherein the immunostimulatory oligonucleotide is a CpG.

In the fourty-first embodiment, the invention provides the vaccinecomposition of any one of thirty-seventh through fourtieth embodiment,wherein said polycationic carrier is present in the amount of betweenabout 0.5 and about 400 mg per dose.

In the fourty-second embodiment, the invention provides the vaccinecomposition of the fourty-first embodiment, wherein said polycationiccarrier is present in the amount of 50-300 mg per dose or 1-25 mg perdose, or 1-10 mg per dose.

In the fourty-third embodiment, the invention provides the vaccinecomposition of any one of thirty-seventh through fourty-secondembodiments, comprising the saponin in the amount of between about 0.1and about 1000 ug per dose.

In the fourty-fourth embodiment, the invention provides the vaccinecomposition of the fourty-third embodiment, wherein the saponin ispresent in the amount of 250-500 ug per dose, or 20-50 ug per dose, or1-10 ug per dose.

In the fourty-fifth embodiment, the invention provides the vaccinecomposition of any one of thirty-seventh through fourty-fourthembodiments, comprising the sterol in the amount of between about 0.1and about 1000 ug per dose.

In the fourty-sixth embodiment, the invention provides the vaccinecomposition of the fourty-fifth embodiment, wherein the sterol ispresent in the amount of 250-500 ug per dose, or 20-50 ug per dose, or1-10 ug per dose.

In the fourty-seventh embodiment, the invention provides the vaccinecomposition of any one of thirty-seventh through fourty-sixthembodiments, comprising the quaternary amine in the amount of betweenabout 1 and about 200 ug per dose.

In the fourty-eighth embodiment, the invention provides the vaccinecomposition of fourty-seventh embodiment, wherein the quaternary amineis present in the amount of about 100 ug per dose or between about 10and about 100 ug per dose or about 5 ug per dose.

In the fourty-ninth embodiment, the invention provides the vaccinecomposition of any one of of thirty-seventh through fourty-eighthembodiments, comprising the immunostimulatory oligonucleotide in theamount of between about 0.5 ug and about 400 ug per dose.

In the fiftieth embodiment, the invention provides the vaccinecomposition of the fourty-ninth embodiment, wherein theimmunostimulatory oligonucleotide is present in the amount of 100-250 ugper dose, or 20-50 ug per dose or about 1 ug per dose.

In the fifty-first embodiment, the invention provides the vaccinecomposition of any one of thirty-seventh through fiftieth embodiments,wherein the oily phase comprises an oil and an oil-soluble emulsifier.

In the fifty-second embodiment, the invention provides the vaccinecomposition of any one of thirty-seventh through fifty-firstembodiments, wherein said oily phase is present in the amount of up to85% v/v.

In the fifty-third embodiment, the invention provides the vaccinecomposition of the fifty-second embodiment, wherein said oily phase ispresent in the amount of 51% v/v.

In the fifty-fourth embodiment, the invention provides the vaccinecomposition of any one of fifty-first through fifty-third embodiments,wherein the oil comprises 40-84% v/v of the vaccine composition, and theoil-soluble emulsifier comprises 1-11% v/v of the vaccine composition.

In the fifty-fifth embodiment, the invention provides the vaccinecomposition of the fifty-third embodiment, wherein the oil comprises 45%v/v of the formulation, and the oil-soluble emulsifier comprises 6% v/vof the formulation.

In the fifty-sixth embodiment, the invention provides a vaccinecomposition comprising an Eimeria maxima or Clostridium perfringensantigen and an adjuvant formulation which comprises:

a) an oily phase, said oily phase being present in the amount of atleast 50% v/v of the composition; a polycationic carrier, andoptionally, an immunostimulatory oligonucleotide; or

b) an oily phase, said oily phase being present in the amount of atleast 50% v/v of the composition; an immunostimulatory oligonucleotide,a sterol, and monophosphoryl lipid A (MPL-A) or an analog thereof.

In the fifty-seventh embodiment, the invention provides the vaccinecomposition of the fifty-sixth embodiment, comprising antigens againstEimeria maxima and Clostridium perfringens.

In the fifty-eighth embodiment, the invention provides the vaccinecomposition of claim the fifty-sixth embodiment or the fifty-seventhembodiment, wherein said polycationic carrier is DEAE-Dextran.

In the fifty-ninth embodiment, the invention provides a use of thevaccine composition according to claims fifty-sixth through fifty-eighthembodiment for treatment or prevention of infections caused by Eimeriamaxima or Clostridium perfringens in poultry.

In the sixtieth embodiment, the invention provides a vaccine compositioncomprising a Neospora antigen and an adjuvant formulation comprising anoily phase, said oily phase being present in the amount of at least 50%v/v of the composition; and

-   -   a) monophosphoryl lipid A (MPL-A) or an analog thereof; or    -   b) a combination of an immunostimulatory oligonucleotide and a        polycationic carrier.

In the sixty-first embodiment, the invention provides the vaccinecomposition of the sixtieth embodiment, comprising the combination ofthe immunostimulatory oligonucleotide and dextran DEAE.

In the sixty-second embodiment, the invention provides the vaccinecomposition of the sixtieth embodiment, comprising monophosphoryl lipidA (MPL-A) or the analog thereof, and further comprising theimmunostimulatory oligonucleotide.

In the sixty-third embodiment, the invention provides the vaccine of thesixty-second embodiment, further comprising a sterol.

In the sixty-fourth embodiment, the invention provides the vaccine ofthe sixty-third embodiment, wherein the sterol is cholesterol.

In the sixty-fifth embodiment, the invention provides the vaccineaccording to any one of sixtieth though sixty-fourth embodiment, whereinthe Neospora antigen is a Neospora caninum antigen.

In the sixty-sixth embodiment, the invention provides use of the vaccineaccording to any one of sixtieth though sixty-fifth embodiment fortreatment or prevention of an infection caused by Neospora.

In the sixty-seventh embodiment, the invention provides a vaccinecomposition comprising a Chlamydophila abortis antigen and an adjuvantformulation comprising an oily phase, said oily phase being present inthe amount of at least 50% v/v of the composition; a sterol; animmunostimulatory oligonucleotide; monophosphoryl lipid A (MPL-A) or ananalog thereof; and poly I:C.

In the sixty-eighth embodiment, the invention provides use of thevaccine according to the sixty-seventh embodiment for treatment orprevention of an abortion caused by C. abortis in ewes.

In the sixthy ninth embodiment, the invention provides a vaccinecomposition comprising myostatin and an adjuvant formulation, saidadjuvant formulation comprising an oily phase, said oily phase beingpresent in the amount of at least 50% v/v of the composition, animmunostimulatory oligonucleotide and either:

-   -   a) a polycationic carrier; or    -   b) MPL-A or an analog thereof.

In the seventieth embodiment, the invention provides the vaccinecomposition of the sixty-ninth embodiment comprising MPL-A or the analogthereof, wherein said formulation contains less than 0.5 ug of a sterolper 50 ul of said composition.

In the seventy-first embodiment, the invention provides the vaccinecomposition of the seventieth embodiment, which contains no sterol.

In the seventy-second embodiment, the invention provides the vaccinecomposition of the seventieth embodiment, wherein the sterol ischolesterol.

In the seventy-third embodiment, the invention provides a use of thevaccine according to any one of embodiments 69 through 72 for loweringan amount of myostatin in an animal.

In the seventy-fourth embodiment, the invention provides the useaccording to the seventy-third embodiment, wherein said animal is apoultry animal.

In the seventy-fifth embodiment, the invention provides a vaccinecomposition comprising an Trueperella pyogenes antigen and an adjuvantformulation, wherein the adjuvant formulation comprises an oily phase,said oily phase being present in the amount of at least 50% v/v of thecomposition; an immunostimulatory oligonucleotide and a polycationiccarrier.

In the seventy-sixth embodiment, the invention provides the vaccinecomposition of the seventy-fifth embodiment, wherein the Trueperellapyogenes antigen is pyolysin.

In the seventy-seventh embodiment, the invention provides the use of thevaccine composition of the seventy-fourth or the seventy-fifthembodiment for treatment or prevention of an infection caused byTrueperella pyogenes.

In the seventy-eighth embodiment, the invention provides a vaccinecomposition comprising an E coli antigen, a BRV antigen or a BCVantigen, and an adjuvant formulation, wherein said adjuvant formulationcomprises an oily phase present in the amount of at least 50% v/v ofsaid vaccine composition, an immunostimulatory oligonucleotide and atleast one of a polycationic carrier and a source of aluminum.

In the seventy-ninth embodiment, the invention provides the vaccinecomposition of the seventy-eighth embodiment, comprising E coli antigen,a BRV antigen and a BCV antigen.

In the eightieth embodiment, the invention provides the vaccinecomposition of the seventy-eighth or seventy-ninth embodiment wherein

-   -   a. E coli antigen, if present, is selected from the group        consisting of E coli K99, E coli F41 and a combination thereof;    -   b. BRV antigen, if present, is selected from the group        consisting of BRV G6, BRV G10 and a combination thereof.

In the eighty-first embodiment, the invention provides the vaccinecomposition according to any one of seventhy-eighth through eightiethembodiment, wherein the polycationic carrier, if present, is dextranDEAE, and the immunostimulatory oligonucleotide is a CpG.

In the eighty-second embodiment, the invention provides the vaccinecomposition according to any one of seventhy-eighth through eighty-firstembodiment, comprising the source of aluminum, which is an aluminumhydroxide gel.

In the eighty-third embodiment, the invention provides the vaccinecomposition of the eighty-second embodiment, wherein said source ofaluminum is present in the amount of 5%-20% v/v.

In the eighty-fourth embodiment, the invention provides the vaccinecomposition of the the eighty-third embodiment, wherein said source ofaluminum is present in the amount of 10%-17% v/v.

In the eighty-fifth embodiment, the invention provides a use of thevaccine composition according to any one of the seventhy-eighth througheighty-fourth embodiment for treatment or prevention of enteritis causedby E coli, BCV or BRV in a bovine animal.

In the eighty-sixth embodiment, the invention provides the use accordingto the ninety-first embodiment, wherein said vaccine causes at least asix-month-long immunity to said antigen(s).

In the eighty-seventh embodiment, the invention provides a vaccinecomposition comprising a Rhipicephalus microplus antigen and anadjuvant, said adjuvant being selected from the group consisting of:

-   -   a) an aqueous adjuvant comprising an immunostimulatory        oligonucleotide, a saponin, a sterol, a quaternary amine, a        polyacrylic polymer, and a glycolipid; and    -   b) an oil-based adjuvant, comprising an oily phase present in        the amount of at least 50% v/v of the vaccine composition and        comprising an immunostimulatory oligonucleotide and a        polycationic carrier.

In the eighty-eigthth embodiment, the invention provides the vaccinecomposition of eighty-seventh embodiment, wherein the saponin is Quil A,the sterol is cholesterol, the quaternary amine is DDA, the glycolipidisN-(2-Deoxy-2-L-leucylamino-b-D-glucopyranosyl)-N-octadecyldodecanoylamideor a salt thereof, and the immunostimulatory oligonucleotide is a CpG.

In the eighty-ninth embodiment, the invention provides the vaccinecomposition of the eighty-seventh embodiment, wherein the polycationiccarrier is dextran DEAE and the immunostimulatory oligonucleotide is aCpG.

In the ninetieth embodiment, the invention provides the vaccinecomposition of any one of the eighty-seventh to eighty-ninth embodiment,wherein the Rhipicephalus microplus antigen is Bm86 protein.

In the ninety-first embodiment, the invention provides a use of thevaccine composition according to any one of the eighty-seventh toninetieth embodiment for treatment or prevention of an infection causedby Rhipicephalus microplus.

In the ninety-second embodiment, the invention provides a vaccinecomposition comprising a Foot-and-Mouth Disease (FMD) antigen and anadjuvant formulation, said adjuvant formulation comprising an oily phasepresent in the amount of at least 36% v/v of said vaccine composition,an immunostimulatory oligonucleotide and a polycationic carrier, whereinsaid vaccine composition is a water-in-oil emulsion. In differentembodiments, said Foot-and-Mouth Disease Virus antigen may be of eitherwild-type FMDV, genetically modified and/or attenuated FMDV strains, orrecombinantly expressed FMDV structural proteins such as virus likeparticles (VLPs) of serotypes A, C, O, Asia1, SAT1, SAT2, or SAT3.

In the ninety-third embodiment, the invention provides the vaccinecomposition of the ninety-second embodiment, wherein theimmunostimulatory oligonucleotide is a CpG, and the polycationic carrieris DEAE dextran.

In ninety-fourth embodiment, the invention provides the vaccinecomposition of the ninety-second or ninety-third embodiment, wherein theantigen is the invention provides the vaccine composition of claim theninety-eighth or ninety-ninth embodiment, wherein the antigen is derivedfrom the genetically modified FMD-LL3B3D platform virus which isattenuated in cattle and pigs, specifically FMD-LL3B3D-A24 Cruzeiro.

In the ninety-fifth embodiment, the invention provides a use of thevaccine composition of any one of the ninety-second or ninety-fourthembodiment for treatment or prevention of FMD in cattle.

In the ninety-sixth embodiment, the invention provides a vaccinecomposition comprising a Streptococcus uberis (S. uberis) antigen and anadjuvant formulation comprising an oily phase, said oily phase beingpresent in the amount of at least 50% v/v of the composition; apolycationic carrier; and

-   -   a) an immunostimulatory oligonucleotide;    -   b) a combination comprising a saponin, a sterol, and a        quaternary amine; or    -   c) a combination thereof.

In the ninety-seventh embodiment, the invention provides a vaccinecomposition of the ninety-sixth embodiment, wherein the antigen is a Suberis adhesion molecule or an immunogenic fragment thereof.

In the ninety-eighth embodiment, the invention provides a use of thevaccine according to any one of ninety sixth or ninety-seventhembodiment for treatment or prevention of an infection caused by Suberis.

The following examples are presented as illustrative embodiments, butshould not be taken as limiting the scope of the invention. Manychanges, variations, modifications, and other uses and applications ofthis invention will be apparent to those skilled in the art.

EXAMPLES Example 1. Development of Recombinant Vaccination Strategy toEnhance Immunity Against Necrotic Enteritis

The aim of the study was to evaluate the effects of in vivo vaccinationwith adjuvanted recombinant clostridia vaccine against live challengeinfection with Eimeria maxima and Clostridium perfringens in NecroticEnteritis Disease Model.

Materials and Methods

Recombinant proteins: Full-length coding sequences for genes encoding C.perfringens (ATCC 13124, American Type Culture Collection, Manassas,Va.) NetB and EF-Tu were cloned by PCR into the pET32a(+) vector with anNH₂-terminal polyhistidine epitope tag. Cloned genes were transformedinto competent Escherichia coli, the bacteria were cultured for 16 h at37° C., and induced for 5 h at 37° C. with 1.0 mM isopropylβ-D-thiogalactopyranoside (Amresco, Cleveland, Ohio). Bacteria wereharvested by centrifugation at 10,000 rpm for 10 min at 4° C.,resuspended in PBS, disrupted by sonication, and centrifuged at 10,000rpm for 15 min. The supernatant was incubated for 1 h at 22° C. withNi-NTA agarose (Qiagen, Valencia, Calif.), the resin was washed withPBS, and purified clostridial proteins were eluted with 250 mM imidazolein PBS, pH 9.2. Protein purity was confirmed on Coomassie blue-stainedSDS-acrylamide gels. Protein concentration was determined using acommercial kit from Sigma.

Animals: One-day-old Broiler birds (Ross/Ross) hatched at theLongeneckers Hatchery (Elizabethtown, Pa.) were be transported to theBARC-East, Building 1082 and the chicks were housed in Petersime starterbrooder units according to the established guidelines of BARC SmallAnimal Care Committee. Birds were kept in brooder pens in anEimeria-free facility and transferred into large hanging cages in aseparate location where they were infected and kept until the end ofexperimental period for the live challenge infection study. Allprocedures regarding transportation, measuring body weight, infection,and collecting blood and spleen were approved by the BARC Small AnimalCare Committee (SOP attached). ARS BARC Small Animal Care Committeeestablished guidelines for animal experiments at BARC and conductsregular inspection of all animal facilities.

Immunization: The primary immunization was performed by subcutaneouslyinjecting one-day old broiler chicks with 100 ul vaccine (Ag 100ug/dose). The secondary immunization was performed by subcutaneouslyinjecting s even-day-old broiler chicks were injected subcutaneouslywith 100 ul vaccine (Ag 100 ug/dose).

Eimeria Challenge: BARC strains of Eimeria spp. which have beenmaintained in the Animal Parasitic Diseases Laboratory and propagatedaccording to the established procedure. E. maxima (41A) was be cleanedby floatation on 5% sodium hypochlorite, washed three times with PBS,and viability enumerated by trypan blue using a hemocytometer. Theoocyst number is based on only sporulated oocysts. Six days afterbooster immunization, chickens were inoculated esophageally with 10,000of E. Maxima using an inoculation needle.

C. perfringens Challenge: Four days after Eimeria infection, birds of NEGroups were inoculated esophageally with 1×10⁹ CFU Clostridiumperfringens each using an inoculation needle.

Analysis: Birds were weighed on the day of arrival, just beforechallenge with EM, before challenge with C. perfringens, 2 days postC.P, and 10 days post C.P. challenge to calculate the weight gain.

For scoring intestinal lesions, birds (5 birds/group) were sacrificedtwo days post C.P. infection. Approximately 20 cm intestinal segmentsextending 10 cm anterior and posterior to diverticulum were obtained andcut longitudinally. Lesion scores were evaluated by 2 independentobservers from 0 to 4 in ascending order of severity of the lesion.

Two major C. perfringens virulence factors in chickens are alphatoxinand the NetB (necrotic enteritis B-like) toxin, both of which areimplicated in the pathogenesis of NE. Additional C. perfringens proteinsthat may be involved in bacterial pathogenesis and host protectiveimmunity including pyruvate: ferredoxin oxidoreductase (PFO) andelongation factor G (EF-G) were previously reported to induce protectiveimmunity against experimental challenge infection with C. perfringens.Accordingly antibody titres to these factors were determined asdescribed below.

Five birds per group were selected at random for blood that wascollected by cardiac puncture immediately following euthanasia. Serawere obtained by low speed centrifugation and used in an enzyme-linkedimmunosorbent assay (ELISA) to measure α-toxin- , NetB-, EF, andPFO-specific antibody levels._Briefly, 96-well microtiter plates werecoated overnight with 1.0 μg/well of purified recombinant α-toxin-,NetB-, EF, and PFO proteins. The plates were washed with PBS containing0.05% Tween (PBS-T) and blocked with PBS containing 1% BSA. Sera (100μl/well) were incubated for 2 hr at room temperature with gentleagitation. The plates were washed with PBS-T, and bound antibody wasdetected with peroxidase-conjugated rabbit anti-chicken IgG (Sigma, St.Louis, Mo.) and peroxidase-specific substrate. Optical density (OD) at450 nm was measured with an automated microplate reader (Bio-Rad,Richmond, Calif.).

Statistical analysis: All values are expressed as mean±SEM. Mean valuesfor body weight gain and lesion score are compared among groups by theTurkey test following ANOVA using SPSS 15.0 for Windows (SPSS Inc.,Chicago, Ill.). Differences among means will be considered significantat p<0.05.

The Experimental Design is illustrated in table 1.

TABLE 1 Infection for NE Group Bird Protein (EM + # (Number) (100μg/bird) Adjuvant CP)* 1 15 − 10 mM Buffer − 2 15 − 10 mM Buffer + 3 15NetB (50 μg) + 10 mM Buffer + EF-Tu (50 μg) 4 15 NetB (50 μg) + 1. TXO +EF-Tu (50 μg) 5 15 NetB (50 μg) + 2. TCMO + EF-Tu (50 μg) 6 15 NetB (50μg) + 3. XO + EF-Tu (50 μg) 7 15 NetB (50 μg) + 4. XOM + EF-Tu (50 μg) 815 NetB (50 μg) + 5. SP-OIL + EF-Tu (50 μg) 9 15 NetB (50 μg) + 6. 5%AMPHIGEN ® + EF-Tu (50 μg) 10 15 NetB (50 μg) + 7. 5% AMPHIGEN ® + +EF-Tu (50 μg) poly I:C 11 15 NetB (50 μg) + 8. 5% AMPHIGEN ® + + EF-Tu(50 μg) CpG 12 15 NetB (50 μg) + 9. 5% AMPHIGEN ® + + EF-Tu (50 μg) DEAEDextran 13 15 10. 5% AMPHIGEN ® + + DDA *Chickens were orally infectedwith 1.0 × 10⁴ oocysts/bird of E. maxima (EM) at day 14 post-hatch andwith 1.0 × 10⁹ CFU/bird of C. perfringens (CP) at day 18.

The compositions of the adjuvants were as follows (per 50 ul):

TXO: SEQ ID NO: 8 was present in the amount of 1 ug, Dextran DEAE waspresent in the amount of 5 ug, light mineral oil was present in theamount of 51% v/v of the composition

TCMO: SEQ ID NO: 8 was present in the amount of 1 ug, cholesterol waspresent in the amount of 1 ug, MPL-A was present in the amount of 1ug/50 ul dose, light mineral oil was present in the amount of 51% v/v ofthe composition

XO: Dextran DEAE was present in the amount of 5 ug, light mineral oilwas present in the amount of 51% v/v of the composition.

XOM: Dextran DEAE was present in the amount of 5 ug, light mineral oilwas present in the amount of 51% v/v of the composition, MPL-A waspresent in the amount of 1 ug.

5% AMPHIGEN®+poly I:C: poly I:C was present in the amount of 1 ug.

5% AMPHIGEN®+CpG: SEQ ID NO: 8 was present in the amount of 1 ug.

5% AMPHIGEN®+DEAE Dextran: DEAE dextran was present in the amount of 25ug

5% AMPHIGEN®+DDA: DDA was present in the amount of 1 ug.

The body weight gain was significantly decreased by EM and CP infectionin the NE control group (P<0.05). However, the body weight gaingenerally increased in the groups immunized with recombinat CP proteins(Net B+EF) by 4^(˜)21%. The significant difference with NE control wasfound in Prot TCMO group which were imunized with CP proteins conjugatedwith TCMO adjuvant.

TABLE 2 Body weight gain Group Treatment Mean SEM 1 Cont 347.93 9.387 2NE cont 286.36 14.436 3 Prot 317.86 7.828 4 Prot TXO 316.42 8.826 5 ProtTCMO 345.73 11.745 6 Prot XO 334.67 8.605 7 Prot XOM 331.17 11.387 8Prot SPO 304.09 10.330 9 Prot AMP 310.09 9.479 10 Prot AMPPIC 314.869.571 11 Prot AMP CPG 313.82 11.976 12 Prot AMP DEAE 299.20 15.000 13Prot AMP DDA 301.25 10.440

TABLE 3 Lesion score Group Mean SEM 2 NE cont 3.0 0.0 3 Prot 2.7 0.2 4Prot TXO 2.5 0.2 5 Prot TCMO 2.6 0.2 6 Prot XO 1.7 0.2 7 Prot XOM 2.40.2 8 Prot SPO 2.4 0.2 9 Prot AMP 1.7 0.1 10 Prot AMPPIC 2.1 0.1 11 ProtAMP CPG 2.3 0.2 12 Prot AMP DEAE 2.1 0.1 13 Prot AMP DDA 2.2 0.2

Six days after EM infection and 2 days after CP infection, serumantibody responses against α-toxin, Net-B, EF, and PFO were evaluated.The results are provided in Table 4. Briefly, CP protein generallyincreased Ab titers against CP antigens in the birds immunized with CPproteins. Ab responses to Net B, EF, and PFO antigens were much higherthan to α-toxin.

TABLE 4 Ab responses to Net B, EF, and PFO antigens

-toxin Net B EF PFO Groups Mean SEM Mean SEM Mean SEM Mean SEM 2 NE cont.36 .02 .33 .01 .21 .01 .22 .01 3 Prot .40 .04 .44 .05 .40 .09 .36 .05 4Prot TXO .39 .03 .41 .04 .56 .05 .40 .03 5 Prot TCMO .34 .02 .42 .03 .48.06 .40 .06 6 Prot XO .34 .02 .53 .05 .38 .07 .36 .04 7 Prot XOM .33 .02.40 .04 .55 .03 .41 .04 8 Prot SPO .30 .01 .33 .02 .19 .02 .20 .02 9Prot AMP .37 .01 .33 .00 .22 .03 .32 .07 10 Prot AMPPIC .32 .02 .53 .06.36 .10 .33 .02 11 Prot AMP .42 .02 .41 .02 .24 .08 .28 .01 CPG 12 ProtAMP .38 .02 .53 .05 .17 .02 .23 .03 DEAE 13 Prot AMP .41 .01 .58 .02 .45.03 .36 .03 DDA

Example 2: Hen Anti-Myostatin Vaccine

Myostatin is a secreted growth differentiation factor that is a memberof the TGF beta protein family that inhibits muscle differentiation andgrowth. Myostatin is produced primarily in skeletal muscle cells,circulates in the blood and acts on muscle tissue, by binding acell-bound receptor called the activin type II receptor. Accordingly,inhibition of myostatin results in animals having an increased amount ofmeat/muscle. One approach to lowering the amount of myostatin in ananimal is to generate an anti-myostatin immune response, which can beconveniently measured by the titers of anti-myostatin antibody. In thisexample, a hen model was used.

Cobb 500 Parent Stock and Ross 308 hens (age 12 to 10 weeks,respectively) were primed with a vaccine containing Myostatin ConjugatedPeptide and an adjuvant formulation. The adjuvant formulations used inthe study are shown in Table 5.

TABLE 5 Treatment groups. Treatment Adjuvant Carrier Dose T01 CFA/IFACRM  50 ug T02 IFA/CFA CRM  50 ug T03 CFA/IFA KLH/CRM  50 ug T04 TCMOKLH/CRM  50 ug T05 TCMO CRM 200 ug/50 ug T06 TMO CRM 200 ug/50 ug T07TCMO CRM  50 ug T08 MO CRM  50 ug T09 TMO CRM  50 ug T10 TXO CRM  50 ugThe designation “200 ug/50 ug” refers to the amount of antigen inprime/boost dose, volume 0.2 ml.

The components in the adjuvants are as described in Table 6.

Cobb 500 Parent Stock and Ross 308 hens were primed on week 12 or 10 andboosted on week 18. The serum titers of anti-myostatin antibody weremeasured by ELISA before the vaccination and every two weeks after theprime until 22 and 20 weeks of age, respectively.

Groups T06, T07, T09 and T10 produced the highest responses (antibodymean geometric titers between 50000 and 15000 on week 22). Among thesefour groups, Cobb 500 birds in Groups T06 and T07 demonstrated meangeometric titers above 100,000.

TABLE 6 Adjuvant Adjuvant Adjuvant Name Components Concentration/doseTCMO SEQ ID NO: 8/Cholesterol/ 10 ug/10 ug/5 ug/Drakeol 5 oil MPLA/oil(45%), SPAN ® 80(6.3%) & TWEEN ® 80 (1.45%) MO (20:80 W:O) MPLA (20:80W:O) low MPLA- 5 ug/Drakeol 6 mineral viscosity emulsion oil, SPAN &TWEEN 80 TMO (20:80 W:O) SEQ ID NO: 8/MPLA (20:80 10 ug/5 ug/Drakeol 6mineral oil, Use MO emulsion and admix W:O) low viscosity SPAN & TWEEN80 CpG and conjugated peptides emulsion TXO SEQ ID NO: 8/DEAE- 10 ug/20ug/Drakeol 5 mineral Dextran oil, (45%), SPAN ® 80(6.3%) & TWEEN ® 80(1.45%)

Example 3. Vaccines Against T. Pyogenes

Truepurella pyogenes (formerly Arcanobacterium pyogenes, and formerlyActinomyces pyogenes and also Corynebacterium pyogenes) often causesevere clinical metritis in cattle characterized by thick, purulentsecretion. The foul odor sometimes associated with this condition isprobably caused by anaerobic bacteria that are also present but notdetected by routine cultural methods. The disease is most frequent indry cows or heifers before or at the time of calving, and occasionallyoccurs in lactating animals as a sequel to teat or udder injury.Economically important diseases caused by this organism includemetritis, and abortion in dairy cows and liver abscesses in feedlotcattle. Pyolysin (PLO), a cholesterol-dependent cytolysin expressed byTruepurella pyogenes, is an important host-protective antigen.

Angus crossbred cattle of approximately 14 months of age were used inthis study. Animals were in overall good health and free of anycomplicating disease at enrollment. Animals had ad libitum access tofeed and water.

Formulations: All bacteria (E. coli and T. pyogenes) at 1×10⁹ per dose.Pyolysin was administered at 150 micrograms per dose to animals ingroups T02-T07. Group T01 was used as a control.

Adjuvant formulations tested in this study were as follows:

-   -   ISC/Poly IC—ISC 1000 μg/Poly I:C 50 μg in a 2 mL dose    -   ISC/CpG—ISC 1000 μg/100 μg CpG (SEQ ID NO: 8) in a 2 mL dose    -   TXO—CpG 100 μg (SEQ ID NO: 8)/DEAE Dextran/Mineral oil 5LT NF in        a 2 mL dose    -   QCDCRT—Quil A 150 μg/cholesterol 150 μg/DDA 100 μg/CARBOPOL®        (polyacrylic polymer) 0.0375%/R1005 1000 μg/CpG (SEQ ID NO: 8)        100 μg in a 2 mL dose    -   QAC—Quil A 500 μg/cholesterol 500 μg/AMPHIGENNIecithin oil        emulsion) 2.5% in a 2 mL dose

Pyolysin antibody was measured using an indirect ELISA, antigen on theplate followed by serum sample (primary antibody) followed byanti-bovine IgG conjugate was measured at days 0, 28, and 56.

All samples and controls were diluted 1:2000, and response determined bycalculation of the ratio of the OD of the sample to the OD of thepositive control (Pos ctrl was a pool of serum from convalescentanimals). Antibody was detected by HRP-conjugated sheep anti-bovine IgG.

TABLE 7 Study design Treatment Number of Dose Uterine Group AnimalsTreatment* Day Dose units Route† Challenge T01 8 Saline 0, 28 2 mL SC,SC Day 56 5 × 10⁸ T02 8 E. coli + T (A). pyogenes + PLO 0, 28 2 mL IN,SC Day 56 in ISC/Poly:IC 5 × 10⁸ T03 8 E. coli + T (A). pyogenes + PLO0, 28 2 mL IN, SC Day 56 in ISC/CpG 5 × 10⁸ T04 8 E. coli + T (A).pyogenes + PLO 0, 28 2 mL SC, SC Day 56 in TXO 5 × 10⁸ T05 8 E. coli + T(A). pyogenes + PLO 0, 28 2 mL IN, SC Day 56 in QCDCRT 5 × 10⁸ T06 8 E.coli + T (A). pyogenes + PLO 0, 28 2 mL SC, SC Day 56 in QAC 5 × 10⁸ T078 PLO in ISC/Poly:IC 0, 28 2 mL IN, SC Day 56 5 × 10⁸ *E. coli strain51323 + T (A). pyogenes strain 51496, PLO = pyolysin †SC = Subcutaneous,IN = Intranasal.

The results are shown in Table 8

TABLE 8 LSM of IgG to PLO (Time Point is Day on Study) Treatment No. Day00 Day 28 Day 56 T01 0.216 0.226 0.208 T02 0.274 0.245 0.444 T03 0.2520.229 0.451 T04 0.205  0.506*  0.590* T05 0.291 0.246 0.373 T06 0.243 0.512*  0.687* T07 0.315 0.280 0.624

Groups T04 and T06 (adjuvants TXO and QAC) performed significantlybetter than control (P<0.05). In addition, multiple trends amongdifferent treatment groups (selected as differences where P<0.1) havebeen found. These trends are summarized in Table 9.

TABLE 9 Differences between the groups on days 0 (first parameter), 28(second parameter), 56 (third parameter). “Y” indicates that P < 0.1.T01 T02 T03 T04 T05 T06 T07 T01 X X X X X X X T02 N, N, Y X X X X X XT03 N, N, Y N, N, N X X X X X T04 N, Y, Y Y, Y, Y N, Y, N X X X X T05 Y,N, Y N, N, N N, N, N Y, Y, N X X X T06 N, Y, Y N, Y, Y N, Y, Y N, N, YN, Y, Y X X T07 Y, N, Y N, N, Y N, N, N Y, Y, N N, N, N Y, Y, Y X

Example 4. Evaluation of Pyolysin Vaccine Formulations in LactatingDairy Cows Against Metritis Challenge

The objective of this study was to evaluate the efficacy of native andrecombinant pyolysin vaccine formulations, adjuvanted with TXO, innon-pregnant lactating Holstein or Holstein cross dairy cows, using anartificial metritis challenge model.

Animals were in overall good health, free of any complicating diseases,and did not receive any chemotherapy, systemic antibiotic or otheranti-inflammatory medication during the seven (7) days preceding andpost vaccination and challenge. They were in their 1^(st) to 3^(rd)parity, had no previous history of metritis, and were not culturepositive for T. pyogenes pre-challenge (day −1 or 0). Animals thatdeveloped clinically significant concurrent disease during the studywere removed.

Animals had ad libitum access to feed for at least 20 hours in each24-hour period, the only exception being when they were milked. A basalcustom blended feed ration, representative of the industry forlactation, was used. Animals were acclimated for at least 7 days priorto the start of the study. The formulated vaccines administered to thecows (n=20 per group) contained the following components: T01—Saline;T02—TXO+native pyolysin (nPLO); T03—TXO+recombinant pyolysin (rPLO).Recombinant pyolysin was obtained by cloning, expression, andpurification of the antigen from Corynebacterium glutamicum. Thepurified protein was then inactivated by treatment with formalin. Nativepyolysin, expressed and purified from Trueperella pyogenes, was alsoinactivated by treatment with formalin. The TXO adjuvant contained CpGoligonucleotides, DEAE-Dextran, mineral oil, and the surfactants Span 80and Tween 80.

On the day of vaccination, the appropriate IVP (Table 10) wasadministered via the subcutaneous route. Vaccine was administered in theneck on Day 0, and on the opposite side of the neck on Day 28. The siteof vaccine administration was evaluated on Study Days 0, 1, 2, 3, 7, 28,29, 30, 31, 35, 49 and 77 for injections site reactions. On the day ofvaccination, site of administration was evaluated to confirm that noswellings were present prior to vaccine administration. On Study Day 28,49 & 77 both sides of the neck were observed. Injection site evaluationswere recorded. Rectal temperature were also measured and recorded onStudy Days 0 (prior to the 1^(st) vaccination), 1, 2, 3, 7, 28 (prior tothe 2^(nd) vaccination), 29, 30, 31 and 35 during the vaccination phase.Rectal temperature were also measured and recorded on Challenge Days 0through 28.

Post-vaccination clinical observations were recorded on Study Days 0, 1,2, 3, 7, 28, 29, 30, 31 and 35 (during the vaccination phase). Inaddition, clinical observations were observed and recorded during theChallenge phase starting on Day 49 through 77.

Antibody responses to pyolysin were determined by ELISA on Study Days 0,28, 49, and last day of the study (d77). A hemolytic inhibition assaywas also performed on each serum sample. This assay measures theanti-pyolysin antibody response, which correlates with the biologicalactivity (protection).

TABLE 10 # of Group Animals Treatment Day Route T01 20 Saline 0, 28 SQT02 20 TXO + Native Pyolysin 0, 28 SQ T03 20 TXO + Recombinant Pyolysin0, 28 SQ

Prior to challenge, the ovarian cycle of all cows was synchronized.Progesterone was administered prior to challenge, and daily throughoutthe 28 day challenge phase. Using a sterile cannula similar to abreeding cannula, 10 mL of an Escherichia coli challenge strain and 10mL of a Trueperella pyogenes challenge strain (predetermined challengedoses), each taken up in a separate syringe, was infused into the uterusof all cows on challenge day 0. To ensure complete delivery of challengematerial, the cannula was flushed out with 10 mL of sterile culturemedia.

Challenge was considered successful if at least 60% of the animals intreatment group T01 (control group) developed metritis. The presence ofmetritis would be indicated by the presence of a mucopurulentuterine/vaginal discharge with a score of 2. (This scoring system wasadopted from the method described by Sheldon et al., Theriogenology,65:1516-1530, 2006; in which scores of 0 and 1 were considered normal.)

The primary variable was the presence of a mucopurulent uterine/vaginaldischarge with a score of ≥2, which indicates the presence of metritis.The uterine/vaginal discharge was collected using an aseptic SimcroMetriCheck™ device with an aseptic cup, and scored beginning onChallenge Day 0 through 28 (study day 49 through 77).

A treatment was considered efficacious if only TO1 cows developedclinical metritis, or if the duration and/or proportion of days ofmucopurulent vaginal discharge (score 2) was significantly shorter(p=<0.1) compared to controls. If there was no significant differencebetween groups for duration and proportion of days with metritis, thenthe frequency of T. pyogenes isolation from the uterine bacterial swabwas used as supportive data for vaccine efficacy. Safety of respectivevaccines was assessed based on injection site evaluations, rectaltemperatures and any adverse effects on lactation.

Metritis data collected (vaginal/uterine discharge present, Yes/No;vaginal/uterine discharge score) was summarized for each animal at eachtime-point, and utilized to determine the frequency distributions ofeach category for each treatment at each time-point. Frequencydistributions of whether an animal was Normal/Abnormal (Normal is ascore=0 or 1; Abnormal is a score≥2) for each metritis sign (e.g.vaginal/uterine discharge score) were summarized by treatment andtime-point. Whether an animal ever had an abnormal (a score≥2) uterinedischarge score was summarized by treatment, using a generalized linearmixed model (Proc Glimmix), with a binomial error distribution and alogit link function. The statistical model included the fixed effect oftreatment, and the random effect of batch. Contrasts were made betweentreatment groups. This was repeated for each metritis variable describedin this paragraph. If Proc Glimmix did not converge for a metritisvariable, then Fisher's Exact Test was utilized instead to comparetreatment groups.

Duration of an abnormal score (for each metritis variable) wasdetermined for each animal, and calculated as “(last time-point abnormalminus first time-point abnormal)+1”. Duration of the abnormal score wasset to zero for animals that had no time-points with an abnormal scorefor that metritis variable. Duration of the abnormal score wascalculated as “(last scheduled time-point of data collection minus firsttime-point abnormal)+1” for animals that were removed from the studyprior to the last scheduled data collection time-point for that metritisvariable. Duration of abnormal score (for each metritis variable) waslog transformed, and then analyzed with a general linear mixed modelwith fixed effect: treatment, and random effect: residual. Linearcombinations of the parameter estimates were used in a priori contrastsafter testing for a significant (P≤0.10) treatment effect. Comparisonswere made between treatments. Back-transformed least squares means,their standard errors and their 90% confidence intervals were calculatedfor each treatment group from least squares parameter estimates obtainedfrom the analyses.

Proportion of days with an abnormal score (for each metritis variable),as well as proportion of days with both a normal E. coli and T. pyogenesabsent from the discharge (absent is considered a value<=1+), weredetermined for each animal. Each was then transformed using the arc sinsquare root transformation prior to analysis. These transformedproportion-of-days variables were then each analyzed with a generallinear mixed model with fixed effect: treatment, and random effect:residual. Linear combinations of the parameter estimates were used in apriori contrasts after testing for a significant (P≤0.10) treatmenteffect. Comparisons were made between treatments. Back-transformed leastsquares means, their standard errors, and their 90% confidence intervalswere calculated for each treatment group from least squares parameterestimates obtained from the analyses. Frequency distributions of whetheran animal had E. coli present (present is considered a value>1+), T.pyogenes present (present is considered a value>1+), and both E. coliand T. pyogenes present (present is considered a value>1+), weresummarized by treatment at each time-point.

Results. The antibody response to pyolysin was assessed by ELISA,measuring serum IgG levels. The results (Table 11), presented as leastsquare mean (LSM) titers, indicate that titers were significantly higherin cows in T02 and T03, versus T01, on study days 28, 49 and 77. Theyalso suggest that there were no statistically significant differencesbetween the titers of groups T02 and T03. With respect to antibodytiters in the uterus, also assessed by ELISA, the results (Table 12)demonstrated that there were significantly higher titers on days 49 and77 in cows within T02 and T03, versus those in T01, on those same days.As for hemolytic-inhibiting antibodies, the results in table 13 indicatethat animals in T02 had significantly higher titers on study days 49 and77 than those in groups T01 and T03.

TABLE 11 LSM¹ of Serum IgG Titers S:P ratio Day 0 Day 28 Day 49 Day 77T01 0.250 ^(a) 0.197 ^(a) 0.178 ^(a) 0.366 ^(a) T02 0.226 ^(a) 0.645^(b) 0.782 ^(b) 0.820 ^(b) T03 0.224 ^(a) 0.626 ^(b) 0.725 ^(b) 0.746^(b) ¹Different superscripts represent significant differences betweengroups.

TABLE 12 LSM¹ of IgG anti-PLO in Uterus Day 49 Day 77 T01 0.033^(a)0.120^(a) T02 0.433^(b) 0.353^(b) T03 0.444^(b) 0.382^(b) ¹Differentsuperscripts represent significant differences between groups.

TABLE 13 LSM¹ of HI Antibody S:P ratio in Serum Day 0 Day 28 Day 49 Day77 T01 0.09 ^(a) 0.10 ^(a) 0.11 ^(a) 0.12 ^(a) T02 0.08 ^(a) 0.78 ^(b)2.75 ^(c) 1.12 ^(c) T03 0.09 ^(a) 0.77 ^(b) 2.13 ^(b) 0.84 ^(b)¹Different superscripts represent significant differences betweengroups.

Regarding the primary variable assessed, the level of mucopurulentuterine/vaginal discharge (Vaginal Discharge Score, or VDS), when theduration of metritis was measured, it was significantly shorter in groupT02, versus groups T01 and T03, as measured at 7 and 10 days followingchallenge with bacteria (Tables 14, 15).

TABLE 14 Duration of Metritis (VDS ≥ 2); LSM¹ Week 1 (days 50 to 56)Challenge Day 0 to 10 T01 4.2^(a  ) 7.1^(a  ) T02 n-PLO 2.2^(b  )4.2^(b  ) T03 r-PLO 4.3^(a,c) 7.4^(a,c) ¹Different superscriptsrepresent significant differences between groups.

TABLE 15 P values; P values; Treatment 1^(st) 7 days 1^(st) 10 daysDifferences T01 v T02 0.0096 0.0276 YES T01 v T03 0.8658 0.6962 NO T02 vT03 0.0063 0.0109 YES

As for the % of days that metritis was evident (i.e. a VDS≥2) within 10days following challenge (Tables 16 and 17), it is evident that groupT02 had fewer abnormal days, compared to groups T01 and T03. Also, itwas demonstrated that T. pyogenes was most frequently isolated from cowsin group T03 (data not shown). Thus, the vaccine effect was mostprominent in group T02 (native pyolysin+TXO).

TABLE 16 % Days Normal Week 1 Challenge Day 0 to 10 T01  70 ± 6.7% 75 ±6.1% (25%) T02 44.3 ± 10.4% 53.1 ± 9.7% (46.9%) T03 67.2 ± 6.4%  73.4 ±5.7% (26.6%)

TABLE 17 P values; 1^(st) P values; 1^(st) 7 days 10 days T01 v T020.0472 0.0594 T01 v T03 0.7712 0.8204 T02 v T03 0.0699 0.0790

An additional study was conducted to evaluate the efficacy ofexperimental metritis vaccines, in novel adjuvant formulations, inpregnant dairy cows. In this study, pregnant cows were vaccinated duringthe dry period. Efficacy was measured during the first 10 days aftercalving (parturition).

Pregnant Holstein or Holstein cross cows, in their 1^(st) through 3^(rd)lactation, were selected for the study. All selected cows were inoverall good health, had no previous history of metritis, and had aknown expected calving date. They were also free of any complicatingdiseases, and did not receive any chemotherapy, systemic antibiotic, orother anti-inflammatory medication during the seven (7) days precedingand following vaccination. Animals that developed clinically significantconcurrent disease at any time during the study were removed. During thecourse of the study, animals had ad libitum access to feed at least 20hours in each 24-hour period, the only exception being during milking.Animals also had ad libitum access to water throughout the study.

The vaccines administered to the groups (n=15/group) were as follows:animals in T01 received a 2 ml vaccine consisting of saline; those inT02 received a 2 ml vaccine consisting of ISCOMS/Poly I:C+nPLO; those inT03 received a 2 ml vaccine consisting of TXO+nPLO; those in T04received a 2 ml vaccine consisting of TXO+Escherichia coli+Trueperellapyogenes+nPLO. (All vaccine antigens were formalin-inactivated.)

Following their arrival, animals were allowed to acclimate for 7 days.Approximately 2 months prior to calving (Study Day 0), animals receivedthe first vaccination, subcutaneously in the left side of the neck,except that animals in group T02 received the vaccine intranasally(Table 18). Twenty-eight days later, all animals received the secondvaccination, subcutaneously in the right side of the neck (Table 18).Beginning with the first vaccination, all cows were dried off.

TABLE 18 # of Group Animals Treatment Day Route T01 15 Saline 0, 28 SC,SC T02 15 ISC + Pyolysin (PLO) 0, 28 IN, SC T03 15 TXO + Pyolysin (PLO)0, 28 SC, SC T04 15 TXO + E. coli + T. pyogenes + 0, 28 SC, SC Pyolysin(PLO)

Beginning on the day of calving, and continuing for 21 days afterwards,the presence of a uterine/vaginal discharge was assessed, and ifpresent, collected and assigned a score, with a score of ≥2 indicatingthe presence of metritis. Approximately 30 mL of blood was collected(Study Days 0, 28, and 49), for determination of antibody responses toE. coli, T. pyogenes, and pyolysin by ELISA. Any adverse reactions, nototherwise captured as part of the procedural data collection, weredocumented.

The primary variable was the presence of a mucopurulent uterine/vaginaldischarge; a score of ≥2 would indicate the presence of metritis. Atreatment was considered efficacious if only T01 cows developed clinicalmetritis, or if the duration of mucopurulent vaginal discharge (score≥2)was significantly shorter (p=<0.1) compared to controls. If present, amucopurulent discharge was collected post-parturition.

Comparisons were made between treatments at each time point. Leastsquares means (back-transformed for serology data), their standarderrors, and their 90% confidence intervals were calculated from leastsquares parameter estimates obtained from the analyses. Ranges andnumber of animals with data were calculated for each treatment group ateach time-point.

Metritis data collected (vaginal/uterine discharge present, Yes/No;vaginal/uterine discharge score; clinical signs) were summarized foreach animal at each time-point, and were utilized to determine thefrequency distributions of each category for each treatment at eachtime-point. Frequency distributions of whether an animal wasNormal/Abnormal (normal is a score=0 or 1; abnormal is a score≥2) foreach metritis sign (e.g. vaginal/uterine discharge score) was summarizedby treatment and time-point. Whether an animal ever had an abnormal (ascore≥2) uterine discharge score was summarized by treatment, andanalyzed using a generalized linear mixed model (Proc Glimmix), with abinomial error distribution and a logit link function. The statisticalmodel included the fixed effect of treatment and the random effects ofbatch, and block within batch. Contrasts were made between treatmentgroups (this was repeated for each metritis variable described in thisparagraph). If Proc Glimmix does not converge for a metritis variable,then Fisher's Exact Test was utilized instead to compare treatmentgroups.

Duration of an abnormal score (for each metritis variable) wasdetermined for each animal, and was calculated as “(last time-pointabnormal minus first time-point abnormal)+1”. Duration of the abnormalscore was set to zero for animals that had no time-points with anabnormal score for that metritis variable. Duration of the abnormalscore was calculated as “(last scheduled time-point of data collectionminus first time-point abnormal)+1” for animals that were removed fromthe study prior to the last scheduled data collection time-point forthat metritis variable. Duration of abnormal score was analyzed with ageneral linear mixed model with fixed effect: treatment, and randomeffects batch, block within batch and the residual. Linear combinationsof the parameter estimates were used in a priori contrasts after testingfor a significant (P≤0.10) treatment effect. Comparisons were madebetween treatments. Least squares means, their standard errors, andtheir 90% confidence intervals, were calculated for each treatment groupfrom least squares parameter estimates obtained from the analyses.

Results. All cows which delivered twins were removed from the study, assuch an event predisposes a cow to metritis, and can skew the data. Cowsremoved included 6 from control group T01, 2 each from groups T02 andT03, and 1 from group T04. Of the remaining cows in each group, theincidence of metritis, and estimated days of metritis were calculated.As can be seen in Table 19, the incidence of metritis in groups T03 andT04 was numerically lower compared to the other groups. The data alsoindicated that groups T03 and T04 had a shorter duration of metritis inthe first 10 days following parturition than did the animals in groupsTO1 and T02. Thus, it can be concluded that native pyolysin, whetheralone or in combination with E. coli and T. pyogenes bacterins, whenadjuvanted with TXO, is effective in reducing the incidence of naturalmetritis in cattle.

TABLE 19 Group Metritis Estimate Days lower 90% upper (# animals)Incidence (%) LSMs CI 90% CI T01 (8) 100 5.2 3.6 17.3 T02 (13) 100 6.75.1 8.7 T03 (13) 84.6 3.7 2.1 6.2 T04 (14) 78.6 3.5 2.0 5.7

Example 5. Mastitis Vaccines in Cattle

E. Coli bacterin J-5 is a known antigen for treatment of mastitis. Inthis study, different adjuvants combined with J-5 bacterins have beenevaluated for anti-mastitis effects.

The study design is summarized in Table 20. Calving occurred on ^(˜)day49. Samples of blood and milk were taken on days zero, 7, 28, 35, 49,63, 70, and 84. The cows were challenged on day 70.

TABLE 20 Treatment Number of Dose Group Animals Treatment Day Dose unitsRoute T01 20 Saline 0, 28 5.0 mL SC T02 20 Escherichia Coli Bacterin,J-5 strain 0, 28 5.0 mL SC (ENVIROCOR ®) T03 20 E. coli TXO 0, 28 5.0 mLSC T04 20 E. coli VACCIMAX ® - CpG 0 2.0 mL SC T05 20 E. coliVACCIMAX ® - Poly I:C 0 2.0 mL SC

The duration of infection caused by E Coli in groups T01-T06 was asfollows: T01—252.1 hrs, T02—213 hrs, T03—191.6 hrs, T04—190.2 hrs,T05—198.7 hrs. The treatments with VACCIMAX®provided the shortestduration of infection. VACCIMAX® is an oil-in-water emulsion comprisingmultilamellar liposomes, wherein the antigen is packaged between thebilayers of the liposomes.

The protective effects of the treatments were also assessed bydetermining the stratified mitigated fraction. The higher is thestratified mitigated fraction, the greater is the protective effect.Again, the formulations with VACCIMAX® had the greatest effect(13.95-17.19 times over control), but the treatment with TXO was alsoeffective (6.24 times over control).

Whole cell serum J-5 specific IgG total antibody responses were measuredusing indirect capture ELISA. The results are summarized in Tables 21and 22.

TABLE 21 stratified mitigated contrast fraction 90% confidence intervalT01 vs T02 2.1 −14.9 to 33.3 T01 vs T03 13.1 −15.4 to 62.9 T01 vs T0430.5    6.4 to 47.9 T01 vs T05 36.1  7.6 to 68

TABLE 22 Time Point Period 0 Period 1 Period 2 Period 3 Period 4 T014996 ^(a) 6787 ^(a) 4457 ^(a) 4049 ^(a) 16303 ^(a) T02 4425 ^(a) 15106^(b) 12498 ^(bc) 20281 ^(c) 51040 ^(c) T03 4815 ^(a) 27806 ^(c) 28982^(d) 27612 ^(c) 49968 ^(c) T04 3465 ^(a) 17969 ^(bc) 7495 ^(ab) 6318^(ab) 22010 ^(ab) T05 4477 ^(a) 18012 ^(bc) 18404 ^(cd) 7805 ^(b) 17626^(ab) Period 0 = at 1st vaccination, 1 = at Day 28, 2 = at Day 49, 3 =prior to challenge, 4 = end of challenge. Treatment groups with the sameletter within each time point are not significantly different at alpha =0.10

Example 6: Neospora Caninum Vaccine

Neospora caninum is a coccidian parasite that was identified as aspecies in 1988. It is an important cause of spontaneous abortion ininfected livestock. In addition to being an important cause of cattleabortions, neosporosis is a significant disease in dogs throughout theworld. If the disease is caught early, dogs may be successfully treatedwith clindamycin and other antiprotozoan drugs. However, the disease isoften fatal to young puppies. Preventative vaccines have been tested oncattle. An inactivated vaccine was made commercially available but hadmixed results. A live vaccine using attenuated N. caninum tachyzoiteshas been more successful but is expensive to produce. In this study, theinventors determined the effects of different adjuvants on theproperties of a vaccine against N. canimum using N. caninum cyclophilin(NcCYP) and profilin (NcPro) as antigens.

Eight to 10 weeks old female BALB/c mice were used for this experiment.All animals were immunized twice at 3 week intervals with rNcCyP andrNcProf in the presence of indicated adjuvant. Three weeks after thesecond immunization, all animals were euthanized and spleen and bloodwere collected. NcCyP/NcProf-specific splenocyte proliferative responsewas determined with a proliferation assay (3 to 4-day).NcCyP/NcProf-specific splenocyte cytokine response was determined bystimulating the splenocytes with Neospora antigen for 48 h and thecytokine levels in supernatant determined by cytokine-specific ELISAs.Serum antibody levels were determined by ELISA. The animals were treatedas summarized in Table 23.

TABLE 23 Antigen (Neospora Amount caninum administered Treat- Amounts(prepared as a 2 ml dose and 1/10^(th) of the cyclophilin to mice at ament Adjuvant 2 mls was used/mouse dose.) (NcCyP) time, ul T01 QCDCRTQuil-A (250 ug/2 ml), Cholesterol (250 ug/2 ml), DDA (100 ug/2 100 (100ug/2 ml), Carbopol (0.075% v/v/2 ml), R1005 ml dose) (1,000 ug/2 ml),CpG (SEQ ID NO: 8; 250 ug/2 ml) T02 TXO CpG (SEQ ID NO: 8; 250 ug/2 ml),DEAE-Dextran NcCyP 100 (100 mg/2 ml), Mineral oil (50% v/v/2 ml), SPAN(100 ug/2 (1.5% v/v/2 ml), TWEEN 80 (7% v/v/2 ml) ml dose) T03 TCMO CpG(SEQ ID NO: 8; 250 ug/2 ml), Mineral oil NcCyP 100 (50% v/v/2 ml), SPAN(1.5% v/v/2 ml), TWEEN 80 (100 ug/2 (7% v/v/2 ml), MPLA (25 ug/2 ml) mldose) T04 QCDCRTc Quil-A (250 ug/2 ml), Cholesterol (250 ug/2 ml), DDANcCyP 100 (100 ug/2 ml), Carbopol (0.075% v/v/2 ml), R1005 (100 ug/2(1,000 ug/2 ml,) with “Tc” = Chimeric-ODN/ORN ml dose) SEQ ID NO: 14(250 ug/2 ml) T05 ISCX ISC = ISCOM (100 ug/2 ml), DEAE-Dextran NcCyP 100(100 mg/2 ml) (100 ug/2 ml dose) T06 Negative Normal saline N/A N/AControl

The properties of the treatment groups above are summarized in Table 24.

TABLE 24 Splenocytes, IFNg production Total IgG, OD IgG2a, OD at IgG1,OD at stimulation by Splenocytes at 1:16000 1:2000 1:2000 index (mean+/− (pg/ml) (mean +/− (mean +/− (mean +/− SEM) (mean +/− SEM) SEM) SEM)SEM) T01 8.0 +/− 7.0 23.0 +/− 15.3  0.2076 +/− 0.6155 +/− 0.3823 +/−(QCDCRT) 0.0547 0.264 0.03145 T02 (TXO) 62.9 +/− 59.0 680.5 +/− 446.7 0.279 +/− 0.6742 +/− 0.7675 +/− 0.06855 0.192 0.08285 T03 (TCMO) 92.1+/− 46.4 961.5 +/− 205.5  0.2722 +/− 0.6217 +/−  0.972 +/− 0.0581 0.33930.199359048 T04 2.2 +/− 0.9 18.1 +/− 15.0 0.10780 +/− 0.2584 +/− 0.4404+/− (QCDCRTc) 0.01125 0.03315 0.0693 T05(ISCX) 5.4 +/− 3.6 84.4 +/− 49.4 0.1313 +/− 0.2255 +/− 0.6486 +/− 0.018 0.0206 0.23585 T06(Control) 1+/− 2 12.0 +/− 7.3   0.0778 +/− 0.18127 +/−  0.22 +/− 0.012 0.00330.00959

Taken together, these data demonstrate superior results obtained usingTXO and TCMO.

Example 7. The Effects of Different Adjuvants on Immune Responses toReproductive Tract Infection with Chlamydophila abortus

C. abortus is an intracellular bacterium causing abortion in sheep andgoats. Infection generally occurs during exposure of naïve ewes toaborted material (e.g., placenta, fluids, fetus). The bacterium bay belatent in infected ewes until breeding and during mid- or lategestation, it is present in placenta and causes necrotizing placentitiseven despite antibody response. After the abortion, ewes are typicallyimmune to reinfection.

It is believed that vaccination can be beneficial before exposure as itprevents the initial infection and prevents homing of the bacteria toplacenta. Higher IFNg associated with the antibody response inpost-abortion immunity is a key correlate of protection. IFNg may alsobe associated with persistence seen in the non-pregnant ewes.

Ewes were vaccinated on days zero and 28 and challenged on day 49.Animals were sacrificed on day 63 and necropsy was conducted. On dayzero, vaginal and whole blood samples were taking for qPCR. Blood wassampled weekly for serology results and on days zero, 7, 28, and 35 forcytokines and Elispot measurements.

Treatment groups are presented in Table 25.

TABLE 25 Group Adjuvant Composition A TCMYO CpG (SEQ ID NO: 8, 100ug/ds), Cholesterol (100 ug/ds), MPLA (100 ug/ds), Poly I:C (50 ug/ds)thickened with 45% mineral oil with 6.3% SPAN ® 80 and QS with TWEEN ®80 (1.45%) and water B TCXMO CpG (SEQ ID NO: 8, 100 ug/ds), Cholesterol(100 ug/ds), MPLA (100 ug/ds), DEAE- Dextran (100 mg/ds), thickened with45% mineral oil with 6.3% SPAN ® 80 and QS with TWEEN ® 80 (1.45%) andwater C TCMO CpG (SEQ ID NO: 8, 100 ug/ds), Cholesterol (100 ug/ds),MPLA (100 ug/ds) thickened with 45% mineral oil with 6.3% SPAN ® 80 andQS with TWEEN ® 80 (1.45%) and water D No adjuvant Normal saline(saline) E No vaccination N/A F No infection or N/A vaccination

The antigen was prepared from an aborted ovine fetus kidney andpropagated on McCoy cells. Elementary bodies were purified bycentrifugation and sonication. The antigen was fixed at 100 ug/dose in0.1% formaldehyde in 0.9% sodium chloride for vaccination.

TABLE 6 Avg. OD on day: Group 0 7 14 28 35 42 49 56 63 A 0.055 0.0900.060 0.192 0.266 0.374 0.314 0.315 0.395 B 0.015 0.052 0.073 0.1370.204 0.234 0.234 0.364 0.460 C 0.057 0.075 0.065 0.217 0.347 0.4940.481 0.487 0.584 D 0.040 0.079 0.074 0.034 0.079 0.078 0.074 0.0440.109 E 0.042 0.082 0.056 0.022 0.038 0.032 0.039 0.008 0.170 F 0.0510.095 0.055 0.016 0.033 0.029 0.038 0.015 0.033

Serology results were obtained using Chek-it ELISA kit and aresummarized in Table 26 above.

IFNg, IL-2 and IL-4 expression levels in Sheep PMBC stimulated withChlamydia AG were determined. The results are in Table 27.

TABLE 27 IFNg, IL-2 and IL-4 expression levels in Sheep PMBC stimulatedwith Chlamydia AG IFNg IL-2 IL-4 Day Day Day Day Day Day Day Day DayGroup 7 28 35 7 28 35 7 28 35 A 18.08 4.20 10.77 4.79 7.62 8.53 3.263.01 1.53 B  1.67 2.05  2.52 4.08 7.35 5.63 2.26 1.12 3.24 C  1.39 1.77 2.61 1.18 1.78 8.09 1.48 0.94 1.09 D  1.58 4.58  2.70 0.87 2.73 3.270.64 1.27 1.24 E  2.52 2.42  2.14 2.53 1.95 1.68 1.44 1.38 1.35 F  0.831.20  2.05 0.74 3.13 3.71 1.91 2.11 1.47

Response of Ovine PBMCs to Chlamydia abortus antigen is summarized inTable 28.

TABLE 28 Response of Ovine PBMCs to Chlamydia abortus antigen Mean SFC ×10⁶ cells Fold Increase Group Day 0 Day 28 Day 35 Day 0 Day 28 Day 35 A20.5 50.0 97.0 1.0 12.1 19.5 B 7.5 38.0 14.0 1.0 26.8 14.0 C 1.0 13.033.5 1.0 12.8 28.8 D 31.0 49.5 33.5 1.0 33.8 33.8 E 15.5 19.5 6.0 1.02.3 0.7 F 10.0 7.0 6.0 1.0 0.8 0.4

In addition, the amount of white blood cells was analyzed (data notshown). A 2-way ANOVA indicates that Group F had significantly higherWBC amount than Group A and B and that Group E had significantly higherWBC amount than Group B.

Nodules at the injection times were also analyzed. As expected, GroupsA-C had bigger nodules than Groups C-D. Among the three adjuvant used(Groups A-C), Group C had the biggest nodule size, followed by Group B,followed by Group A.

The volume of nodules was determined. Again, groups A-C had greaternodule volumes than group D-F. Among groups A-C, Group A had thesmallest volume. The nodules in Groups A and B had more hemorrhagingand/or nectrotic tissue. The nodules in Group C had more fibrosis.Cellular characteristics are similar in all three nodules, though GroupC may have more lymphocytic component.

Example 8. Addition of Aluminum to TXO Results in an Improved Stability

The current TXO blend formulation contains 50 mg/ml of DEAE Dextran.Dextran, when present at high concentrations in subcutaneous injections,can cause injection site reactions in the animals. Hence it is proposedto try varying concentrations for DEAE Dextran to check if safety andgood therapeutic value can be obtained without compromising thestability of the vaccine formulation.

Characterization and stability tests are important as they inform uswhether this vaccine can be formulated consistently and with a goodshelf life for manufacturing. Viscosity tests are performed at a rangeof shear rates in order to look for shear thinning (drop in apparentviscosity as shear rate goes up) or shear thickening (increase inapparent viscosity as shear rate goes up), which is a flowcharacteristic of Non-Newtonian fluids. Syringe force tests areperformed to ensure that the vaccine will be easy to draw out and easyto administer over a large number of doses in the field.

Since the immunostimulating oligonucleotide is not expected to alter thestability of the formulation, it was not added to the adjuvant mixturesin this example. AXO (Aluminum+Dextran+Oil) blends of varyingREHYDRAGEL® (5% to 16%) and DEAE Dextran (50 mg/ml-10 mg/ml)concentrations are formulated tested for viscosity, syringe force andsettling using an XO (Dextran+Oil) blend as a control. The testedcompositions were as follows:

Approximately 10 ml of sample was filled into each of five 15-ml Corningcentrifuge tubes and left still over a week in order to observe anaccelerated settling effect on the emulsions due to the tubes' narrowdimensions and conical bottom. The samples were also tested forsyringeability and viscosity. The results are shown below.

TABLE 29 Aqueous Phase REHYDRA- GEL ® 10 Organic phase DEAE 2% w/v mMMineral LOT Dextran Al₂O₃ TWEEN80 PBS Oil SPAN 80 124008- 50 mg/ 0 1.45%v/v q.s 45% v/v 6.3% v/v 65 ml 124008- 50 mg/  5% v/v 1.45% v/v q.s 45%v/v 6.3% v/v 95 ml 124008- 20 mg/ 10% v/v 1.45% v/v q.s 45% v/v 6.3% v/v83 ml 124008- 10 mg/ 16% v/v 1.45% v/v q.s 45% v/v 6.3% v/v 89 ml

These data indicate that upon subjection to accelerated settling in thecentrifuge tubes, the blend with 16% REHYDRAGEL® is the most stable.Further, from previous work by the inventors, it was known that higherDEAE Dextran concentration is associated with higher viscosities andpossible shear thinning. The results of these experiments indicate thatthe addition of REHYDRAGEL® more than compensates for anticipated lossin the shear thinning (pseudoplastic) properties afforded by DEAEDextran. It was also observed that even though the 16% REHYDRAGEL®formulation had a higher syringe force, it was not noticeably harder toinject (syringe force for water is 3N).

TABLE 30 Viscocity Syringe Force Lot (cP) (Newtons) Settlingobservations (Day 7) 124008-65 180 6.5 Thin layer of aq. Phase observedat top 124008-95 160 6.5 Slight settling observed 124008-83 180 6.5 Thinlayer of aq. Phase observed at top 124008-89 180 7.5 No Settlingobserved

From the overall data, it is apparent that the blend with 16%REHYDRAGEL® and 10 mg/ml DEAE Dextran is optimal for use in vaccineformulations, particularly those requiring binding of free endotoxinand/or where longer emulsion shelf-life may be desired.

Example 9. BRV, BCV, and E coli Antigens

In this example, the inventors research the use of adjuvants of theinstant invention in vaccines against enteritis. Enteritis is caused bybacterial, viral and/or parasitic infections. Cattle, particularly,newborn dairy and beef calves are vulnerable to calf scours because theyare subject to many stresses during the first few hours of life whentheir immune systems aren't fully developed. Fluid loss due to calfscours results in dehydration and often, death. Animals that survivecalf scours often remain weak and perform poorly throughout their lives.Agents associated with scours include bacteria, particularly E coli K99and F41, and viruses, such as Bovine Coronavirus (BCV) and BovineRotavirus (BRV).

Ten-month old Holstein steers were used in this study. The animals wereseronegative or low tittered for E coli (K99 and F41), BRV (B223 andLincoln) and BCV.

Treatment groups were as follows:

TABLE 31 Amounts Volume Group N Antigen Adjuvant per dose Rt (ml) T01 10Saline N/A SQ 2 T02 10 ROTAVEC ® Mineral Oil + Alum NA (Commercial IM 2(E. coli K99, BRV product) G6 and BCV T03 10 E. coli K99/F41; Quil A +Cholesterol + Quil-A (500 ug/2.5 ml SQ 2.5 BRV G6/G10, REHYDRAGEL ® (15%dose), Cholesterol BCV (all v/v, 2% Al₂O₃ w/v) + (200 ug/2.5 ml dose),inactivated) CpG (SEQ ID NO: 8) REHYDRAGEL ® (15% v/v), CpG (100 ug/2.5dose ml) T04 10 Quil A + Cholesterol + Quil-A (500 ug/2.5 ml SQ 2.5REHYDRAGEL ® (15% dose), Cholesterol v/v, 2% Al₂O₃ w/v) + (200 ug/2.5 mldose), CpG (SEQ ID NO: 8) + REHYDRAGEL ® AMPHIGEN ® (15% v/v), CpG (100ug/2.5 ml dose) , Amphigen (2.5% v/v) T05 10 TXO + REHYDRAGEL ® CpG (100ug/5 ml SQ 5 (15% v/v, 2% Al₂O₃ dose), DEAE-Dextran w/v) (100 mg/5 mldose), Mineral Oil (45% v/v), Span (6.3%), Tween (1.45% v/v) T06 10 TO +REHYDRAGEL ® CpG (100 ug/5 m1 SQ 5 (15% v/v, 2% Al₂O₃ dose), Mineral Oilw/v) (45% v/v), Span (6.3%), Tween (1.45% v/v), REHYDRAGEL ® (15%)

Blood samples were collected every 21 days for six months for serology.Injection site reactions were measured at Days 0 (pre-vaccination), 1,2, 3, 7, 14, 21 and every 21 days thereafter. Responses to E coli K96, Ecoli F41, BRV Lincoln, BRV B223 and BCV were measured by quantifyingantibody titers on selected days. The results are summarized below(different letters indicate differences at α=0.1):

TABLE 32 Mean geometric titers against viruses (LSM) BRV G6 (Lincoln)BRV G10 (13223), Target titer > 1255 Target titer > 1472 BCV, Targettiter > 1107 Day 0 Day 21 Day 189 Day 0 Day 21 Day 189 Day 0 Day 21 Day189 T01 142.1 ^(b)  208.1 ^(a)  152.3 ^(a) 430.5 ^(a)  512.1^(a)  588.5^(a) 238.9^(a)   430.6 ^(a)  548.8 ^(a) T02 129.2 ^(b)  750.5 ^(b) 349.9 ^(b) 530.1 ^(a) 1024.2^(b)  675.7 ^(ab) 349.8 ^(b)  5997.1 ^(b)1398.9 ^(b) T03 140.2 ^(b) 3649.6 ^(c)  533.9 ^(b) 532.2 ^(a) 2681.9^(c) 985.6 ^(ab) 348.4 ^(ab)  7299.1 ^(bc) 1106.1 ^(b) T04  91.4 ^(ab)3128.8 ^(c)  575.0 ^(b) 492.7 ^(a) 3511.8^(cd) 1064.3 ^(b) 298.8 ^(ab) 7023.0 ^(bc) 1448.3 ^(b) T05  81.8 ^(ab) 4705.6 ^(c) 1489.9 ^(c) 494.6^(a) 9089.6^(e) 2998.6 ^(c) 326.3 ^(ab) 10085.5 ^(c) 2521.6 ^(c) T06 39.1 ^(a) 2682.1 ^(c) 4706.4 ^(a) 456.2 ^(a) 6020.2^(de) 4683.8 ^(c)237.2 ^(a)  9556.2 ^(c) 2298.9 ^(c)

Treatments T05 and T06 resulted in the highest antibody titers from day21 until the end of the study (day 189). Notably, commercial vaccine(ROTAVEC®) did not perform as well as T05 and T06 in inducing antibodiesagainst the viral components of enteritis.

TABLE 33 Mean geometric titer of anti-E. coli E. coli K99 pilus antigen(Target > 742) E. coli F41 pilus antigen (Target undetermined) Group Day0 Day 21 Day 106 Day 189 Day 0 Day 21 Day 106 Day 189 T01 35 ^(a)   36^(a)   82 ^(a)   44 ^(a) 187 ^(a)   152 ^(a)  142 ^(a)   66 ^(a) T02 41^(a)  349 ^(b) 4386 ^(c) 2986 ^(c) 152 ^(a) 12801 ^(c) 6970 ^(d) 4223^(e) T03 43 ^(a)  588 ^(bc)  686 ^(b)  467 ^(b) 200 ^(a)  3200 ^(b)  467^(b)  200 ^(b) T04 50 ^(a)  588 ^(bc) 1089 ^(b)  686 ^(b) 147 ^(a)  3734^(b)  800 ^(b)  400 ^(cd) T05 50 ^(a) 1056 ^(d) 3200 ^(c) 2986 ^(c) 264^(a) 12801 ^(c) 1600 ^(c)  607 ^(d) T06 54 ^(a)  864 ^(cd) 3456 ^(c)1601 ^(c) 216 ^(a)  1600 ^(b)  800 ^(b)  234 ^(bc)

Treatments T02 and T05-T06 performed similarly well in elicitingresponse against K 99. Treatment T02 elicited the best response to Ecoli F41. Treatment T05 was the second most effective in elicitingresponse to that antigen.

Taken together, these data demonstrate that T05 and T06 appear to be themost promising formulations. Both have delivered target IgG responsesfor multiple fractions through day 189. Both T05 and T06 appear toprovide superior or equivalent serological efficacy compared to ROTAVEC®(T02, IM) by SQ administration. T03 and T04 retained elevatedserological titers for the shorter duration than T05 and T06. With asingle dose vaccination T03, T04, T05 and T06 delivered above the targetlevel serum titers for BRV G6, BRV G10 and BCV. With a single dosevaccination T04, T05 and T06 delivered above the target level serumtiters for E. coli K99. T04, T05 and T06 retained anti-virus serumtiters above the target levels for 6 months. T05 and T06 retainedanti-E. coli K99 serum titers above the target levels for 6 months. Allformulations evaluated have demonstrated adequate safety in Holsteinsteers.

Rectal temperatures were measured on days zero, 1, 2, and 3. While therewere statistically significant differences between group T01 (control)and groups T02-T06, the differences in temperatures (LSM) were not great(within one degree F.).

TABLE 34 * Day 000 Day 001 Day 002 Day 003 T01 101.1 ^(a) (38.4) 102.2^(ab) (39.0) 102.0 ^(a) (38.9) 102.3 ^(b) (39.1) T02 101.7 ^(b) (38.7)102.6 ^(abc) (39.2) 102.2 ^(ab) (39.0) 101.8 ^(a) (38.8) T03 101.8 ^(b)(38.8) 102.7 ^(bc) (39.3) 102.2 ^(ab) (39.0) 101.9 ^(a) (38.8) T04 101.3^(ab) (38.5) 102.8 ^(c) (39.3) 102.0 ^(a) (38.9) 102.0 ^(ab) (38.9) T05101.7 ^(b) (38.7) 102.3 ^(abc) (39.1) 102.5 ^(b) (39.2) 102.4 ^(b)(39.1) T06 101.6 ^(ab) (38.7) 102.1 ^(a) (38.9) 102.4 ^(b) (39.1) 102.1^(ab) (38.9)

Preliminary testing of formulations in pregnant dairy cows hasdemonstrated safety. Groups T01, T03 and T05 were tested, 5 cows in eachgroup. Thirteen of 15 cows have calved, 12 calves were normal, one wasstillborn.

Example 10. Anti-Tick Vaccine Experiment Design

Two vaccine formulations based on the Bm86 antigen were tested. Oneformulation contained an aqueous adjuvant (QCDCRT) and the other anoil-based adjuvant (TXO), as summarized below.

TABLE 35 Adjuvant (80% Group Antigen volume) Amounts of ingredients T01N/A None N/A T02 Rhipicephalus microplus QCDCRT 250 ug Quil-A, 250 ugCholesterol, 100 ug (formerly Boophilus) DDA, 0.0375% Carbopol, 1,000 ugR1005, purified rBm86 protein 100 ug SEQ ID NO: 8 T03 (stock, 1.16mg/ml) TXO 100 ug SEQ ID NO: 8/100 mg DEAE- Dextran in mineral oil(45%), SPAN ® 80(6%) & TWEEN ® 80 (1.45%)

Since the adjuvants were used at 80% volume, as described above, onedose of the vaccine composition administered to group T03 contained 100ug SEQ ID NO: 8/100 mg DEAE-Dextran, 36% v/v of mineral oil, 4.8% v/vSPAN® 80, and 1.16% v/v TWEEN® 80. Since there was no oil in QCDCRT, theconcentrations of the ingredients were the same as in Table 35.

Twenty-four calves were randomly assigned into one of three treatmentgroups of eight calves each. The calves of each treatment group wereindividually vaccinated with 2 cc of either one of the two Bm86+adjuvantformulations or saline (control group). Vaccinations occurred on day 0and 28. On day 41, the cattle were placed into stanchion, and on day 42were infested with 250 mg of R. annulatus larvae. The ticks used in thisstudy were originally collected from a ranch in Val Verde County, Texas.All detaching engorged adult females were collected daily fromindividual calves on days 63-84. Calves were removed from stanchion onday 85. Collected ticks were counted and up to 10 from each calf wereweighed and placed into an environmental chamber each day of collectionfor 13 days. Spent females were discarded 14 days after collection andthe egg mass produced weighed. Fourteen days after the first hatch, thenumbers of hatched and intact eggs were recorded and a determination ofpercent hatch was calculated. Before each injection with vaccine and forthe following three days post injection, the injection site wasmonitored on each calf for lumps, and rectal temperatures were taken.Blood serum was collected from each calf on days −7, 0, 14, 28, 42, and85 for the determination of Bm86 antibody titers throughout the study asdetermined by ELISA.

Results

Preliminary results show 98.6 and 99.6% control from T02 and T03formulations, respectively, which is significantly higher than T01.These percent control calculations take into account only the reductionin engorged females and egg mass weight. Reduction in percent hatch willbe determined at a later date and added to the final results. One of the24 calves in the study produces a small lump after each injection(formulation containing the oil adjuvant). The lumps are less than 10 cmin length and 3 cm in depth. Lumps are soft and do not seem painful tothe animal. There are no increases in rectal temperature from thetreated animals throughout the study.

Serology results demonstrate statistically significant differencesbetween each of the treatments on the respective time points exceptthere was no statistical significance (p=0.114) between treatments withQCDCRT and TXO at 14 days time point. Both QCDCRT and TXO effectivelyincrease BM86 antibody titers at each time point tested. TXO wassuperior to QCDCRT (p<0.05) at each time point tested except on day 14(p=0.114).

TABLE 36 BM86 antibody titer, Back Transformed Least Square mean (Mean ±SEM) Group N Day 14 Day 28 Day 41 Day 83 T01 8   100 ± 46.42   100 ±31.52   100 ± 17.05   100 ± 17.31 T02 8 2018 ± 937  1179 ± 372  13532 ±2308 4082 ± 707 T03 8 5956 ± 2765 8404 ± 2649 28557 ± 4870 18638 ± 3227

Example 11. Foot-and-Mouth Disease (Guinea Pigs)

The goal of this study was to compare the humoral immune responses inguinea pigs vaccinated with trivalent FMD vaccines adjuvanted withdifferent adjuvant formulations. Guinea pigs were vaccinated on dayszero and 28 as summarized in Table 37.

In each dose of T03-T07, the antigen was a combination of FMVD Type 0 (9ug), A (5 ug) and Asia1 (5 ug/ml). Antigen composition in T02 isproprietary information of the manufacturer and thus was unavailable.

Blood samples were collected for serology study on days −3, 25, and 53.Serum titers of antibodies against serotypes 0, A, and Asia 1 aresummarized below.

While the responses against Sero types 0 and A were low even in positivecontrol group (T02), the response against Asia 1 were higher in T07 (TXOadjuvant) than in the positive control group, and greater than in anyother treatment. The low responses against serotypes 0 and A may be dieto presence of low levels of O and A antigens in the formulation.

Notably, liposome-based VACCIMAX® groups (T03-T05) did not demonstratesignificant response against any of the antigens (O, A, Asia1).

TABLE 37 Treatment Group N Adjuvant/Ingredients days Dose RT T01 24/22Saline 0, 28 0.2 IM ml T02 24/24 Commercial Proprietary 0, 28 0.2 IMvaccine-Raksha ml Monovalent FMDV vaccine (vet) (positive control) T0323/22 VACCIMAX ® S100/Cholesterol, 12% w/v 0, 28 0.2 IM S100/polyl: C(60 mg/dose)/Poly I: C 100 μg/dose ml Water in Oil, 50% aqueousliposomes + 50% Marcol 52/ Montanide 888 T04 23/23 VACCIMAX ®Proprietary 0, 28 0.2 IM S100/Pam3Cys ml T05 23/22 VACCIMAX ® VacciMaxBiolipon 95, 100 ug/Poly 0, 28 0.2 IM (Biolipon 95)/ I: C/ds, Water inOil, 50% aqueous ml Pam3Cys liposomes + 50% Marcol 52/ Montanide 888 T0624/24 QCDCRT (80% 250ug Quil-A, 250 ug Cholesterol, 0, 28 0.2 IM volume)100 ug DDA, 0.0375% Carbopol, ml 1,000 ug R1005, 100 ug SEQ ID NO: 8 T0724/23 TXO (80% 100 ug SEQ ID NO: 8; 100 mg 0, 28 0.2 IM volume) DextranDEAE, 45% mineral oil, ml 6.3% SPAN ®80, 1.45% TWEEN ®80, QS water Nrepresents the number of animals surviving for the first/secondvaccination

Since adjuvants TXO and QCDCRT were used at 80% volume, as describedabove, one dose of the vaccine composition administered to group T07contained 100 ug SEQ ID NO: 8/100 mg DEAE-Dextran, 36% v/v of mineraloil, 5.04% v/v SPAN® 80, and 1.16% v/v TWEEN® 80. Since there was no oilin QCDCRT, the concentrations of the ingredients were the same as inTable 37.

TABLE 38 Sero Type O Sero Type A Sero Type Asia1 SN titers, Geonetric SNtiters, Geonetric SN titers, Geonetric mean titer mean titer mean titerDay Day Day Day Day Day Day Day Day Group −3 28 56 −3 28 56 −3 28 56 T014 4 4.1 4 4 4 4 4.1 4.1 T02 4 9.6 49.9 4 7 14.1 4 14 266.9 T03 4 4.813.8 4 4.1 9 4 5.9 24.3 T04 4 4.1 10.3 4 4.5 5.8 4 4.3 9.7 T05 4 4.1 8.14 4.5 4.7 4 4.3 13.6 T06 4 4.1 5 4 4 4 4 4.2 5.1 T07 4 5 9 4 5.6 43.1 419.8 320.8

Example 12. Foot-and-Mouth Disease (Cattle)

In this study, the effect of different adjuvants used in a vaccineagainst FMD in a challenge model was determined. Three adjuvants werestudied. The vaccine was an ARS experimental vaccine against FMD in achallenge model developed by PIADC. FMD-LL3B3D-A24 Cruzeiro was usedboth as the antigenic component of the vaccine (10 ug) and wild typeFMDV A-24 Cruzeiro was used as the challenge virus. The antigen waspreviously described e.g., in US20120315295 (Rieder at al, filed on Jun.9, 2011 and published on Dec. 13, 2012). Briefly, FMD-LL3B3D-A24Cruzeiro comprises a genetically modified FMDV (Foot-And-Mouth-DiseaseVirus). The FMDV is genetically modified, i.e., it is a leaderless viruscontaining a deletion of the leader (L_(pro)) protein coding region suchthat FMD viruses lacking this protein are attenuated in cattle and pigs.It also comprises mutations (negative markers) introduced in twonon-structural viral proteins resulting in the elimination of twoantigenic epitopes recognized by specific antibodies, one located inprotein 3B and the other in protein 3D (replaced by the correspondingsequence of bovine rhinovirus that serves as negative antigenic epitopein these proteins), thus providing two possible targets for DIVA(Differentiation of naturally Infected from Vaccinated Animals)serological tests.

Four to seven bovines were used in each group. Total volume injected was2 ml. Animals were vaccinated on day zero by an IM injection (2.0 ml perdose) and challenged on day 21 by intra-dermal route with wild typeFMDV. Clinical scores were assessed on days 0, 3, 7, and 10 according tothe following scale: no clinical signs: 0, vesicular foot lesions: 1point for each foot affected. Maximum score is 4. The results of theexperiment are as follows:

TABLE 39 Average clinical score Group Adjuvant Details/Per dose Day 0Day 3 Day 7 Day 10 T01 Saline N/A 0 3.0 3.5 4.0 T02 MONTANIDE ® Amineral oil based adjuvant which has 0 0 0 0 ISA 206 VG been developedfor the manufacture of Water-in-Oil-in-Water (W/O/W) emulsions. Itcomprises a high grade injectable mineral oil and an extremely refinedemulsifier obtained from mannitol and purified oleic acid of vegetableorigin. MONTANID ®E ISA 206 VG is free of animal origin ingredients. Theexact composition is proprietary to manufacturer (Seppic Inc) T03 QCDCRT(80% 250 ug Quil-A, 250 ug Cholesterol, 0 1.14 2.86 2.43 volume) 100 ugDDA, 0.0375% Carbopol, 1,000 ug R1005, 100 ug SEQ ID NO: 8 T04 TXO (80%100 ug SEQ ID NO: 8/100 mg DEAE- 0 0 0 0 volume) Dextran in WO emulsion

Differences between T01 and T02 and between T01 and T04 werestatistically significant.

From the table above, it can be concluded that at least based on theclinical score, adjuvants TXO and MONTANIDE®ISA 206 VG are about equallyefficient. However, serology analysis to measure serum neutralizingactivity against FMDV-A24 demonstrates that group T04 (adjuvant TXO) hadhigher titers than group T02 (MONTANIDE®ISA 206 VG).

TABLE 40 Serum Neutralization titers Least Square Means(back-transformed) Time point Day 0 Day 7 Day 14 Day 21 T01 0.45^(a)0.45^(a) 0.45^(a) 0.45^(a) T02 0.45^(a) 1.60^(c) 1.13^(c) 1.20^(b) T030.45^(a) 1.10^(b) 0.66^(b) 0.61^(a) T04 0.45^(a) 2.21^(d) 2.21^(d)2.21^(d) Diferent letters indicate statistically significant difference(p <= 0.05)

These results demonstrate that TXO adjuvant was able to provide 100%protection against a challenge with a FMD causing agent in cattle andconfer higher antibody titers than the saline control and the other twoadjuvants tested.

The amount of FMDV RNA (copies per mL) was determined in nasal swabs andin serum. The data are provided in tables 41-44. Briefly, these datademonstrate that groups T02 and T04 resulted in lower amounts of FMDV innasal swabs. Among T02 and T04, it is noted that animals in group T04demonstrated earlier decrease (or lack of increase) in FMDV RNA amounts,thus again demonstrating superior properties of adjuvant TXO.

TABLE 41 FMDV in nasal swabs (“Shedding” FMDV RNA copy per ml measuredby rRT-PCR; LS means) Time Point Day Day Day Day Day Day Day Day Day 21Day 22 Day 23 24 25 26 27 28 29 30 31 T01 1.35 1.75 6.16 7.09 6.88 6.135.19 4.98 2.51 1.81 1.35 T02 1.35 1.49 4.63 5.31 5.62 4.14 2.21 1.841.58 2.66 1.83 T03 1.35 1.92 3.54 4.66 5.39 5.27 3.12 1.87 1.56 1.841.84 T04 1.35 1.59 3.97 5.05 4.47 3.75 2.31 1.61 1.56 2.58 1.55

TABLE 42 Statistical significance (nasal swabs) P< = 0.05? Day Day DayDay Day Day Day Day Day Day Day 21 22 23 24 25 26 27 28 29 30 31 T01 vsT02 No No Yes Yes No Yes Yes Yes No No No T01 vs T03 No No Yes Yes No NoYes Yes No No No T01 vs T04 No No Yes Yes Yes Yes Yes Yes No No No T02vs T03 No No No No No No No No No No No T02 vs T04 No No No No Yes No NoNo No No No T03 vs T04 No No No No No Yes No No No No No

TABLE 43 FMDV in serum (“Viremia”, FMDV RNA copy number per ml measuredby rRT-PCR; Time Point Day Day Day Day Day Day Day Day Day Day 21 Day 2223 24 25 26 27 28 29 30 31 T01 1.35 6.85 8.67 8.56 5.96 3.91 1.77 1.351.35 1.35 1.35 T02 1.35 1.58 1.57 1.75 1.83 1.35 1.35 1.35 1.35 1.351.35 T03 1.35 3.84 3.77 3.20 2.72 1.82 1.35 1.35 1.35 1.35 1.35 T04 1.351.59 1.35 1.35 1.35 1.35 1.35 1.35 1.35 1.35 1.35

TABLE 44 Statistical significance (serum) P< = 0.05?+0 Day Day Day DayDay Day Day Day Day Day Day 21 22 23 24 25 26 27 28 29 30 31 T01 vs NoYes Yes Yes Yes Yes Yes No No No No T02 T01 vs No Yes Yes Yes Yes Yes NoNo No No No T03 T01 vs No Yes Yes Yes Yes Yes Yes No No No No T04 T02 vsNo Yes Yes Yes No No No No No No No T03 T02 vs No No No Yes Yes No No NoNo No No T04 T03 vs No Yes Yes Yes Yes No No No No No No T04

While all animals in group TO1 exhibited fever after the challenge, noneof animals in group T04 had fever. The responses in groups T02 and T03were inconsistent (some animals exhibited fever and some did not). Thisobservation confirms the conclusion of general superiority of TXOcompared to the other adjuvants used in this study.

Example 13: TXO Activates Cell-Mediated Immunity

Using FMD as an exemplary antigen in the animal model described in theprevious example, the effect of the adjuvants on cell-mediated immunitywas analyzed. Peripheral blood mononuclear cells (PBMC) were purifiedfrom bovine whole blood collected on days 4, 7, 14, and 21post-vaccination. FMDV-specific T cell proliferative responses wereassessed using Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE)staining.

The results are provided in table 45.

TABLE 45 Proliferation index (Mean ± SD) Treatment/DAY Day 4 Day 7 Day14 Day 21 T01 1 ± 0  1.22 ± 0.365  1.12 ± 0.070 0.965 ± 0.144 T02 1 ± 01.633 ± 1.046 1.813 ± 0.860 4.473 ± 4.012 T03 1 ± 0 1.769 ± 0.877 1.850± .980  1.549 ± 0.608 T04 1 ± 0 1.589 ± 0.682 2.667 ± 1.424 6.757 ±4.653

These data demonstrate superior effect of TXO on cell-mediated immunityboth at day 14 and day 21. These data also indicate that sincecell-mediated immunity is responsible for duration off immunity,adjuvant TXO can possibly provide a longer duration of immunity thanMONTANIDE®ISA 206 VG.

Example 14. Generation of Antibodies for Diagnostic Use

Adjuvant TXO of the instant invention was used to generate antibodiesfor diagnostic use. Briefly, source animals have been immunized every2-4 weeks with formulations comprising selected recombinant antigensadjuvanted with TXO, the composition of which was as follows: SEQ ID NO:8-125 ug; DEAE dextran—125 mg; Mineral oil—46.56% v/v of theformulation; TWEEN® 80-1.5% v/v of the formulation; SPAN® 80-6.518% v/vof the formulation.

The adjuvant formulation was used at 80% v/v of the vaccine composition.Accordingly, the final concentration of the components of adjuvantformulations was as follows: SEQ ID NO: 8-100 ug; DEAE dextran—100 mg;Mineral oil—37.248% v/v of the vaccine composition; TWEEN® 80 —1.2% v/vof the vaccine composition; SPAN® 80-5.214% v/v of the vaccinecomposition. The final volume was 2 ml.

Small visible injection site reactions were observed after injections,but were within the anticipated size of reactions. Based on dailyobservations, the reactions observed over the ribs did not appear to bepainful to the goats.

Blood samples were collected 2-3 weeks after each immunization, andvarious assays were run to evaluate antibody titer. Serology ELISA titerover 1000 was considered sufficient to start antibody harvesting.

The animals were bled weekly (7.5% of the blood volume based on bodymass). At the conclusion of the study, goats were euthanized by terminalexsanguinations and that blood was also collected for antibodyharvesting. If necessary or the animals were repurposed for additionalstudies.

TABLE 46 Summary of Completed Goat Polyclonal Reagent Generation StudiesTotal Immu- (ug)/dose Source Serum niza- Amount Animal Source Volumetions, Antigen (N) of ABs Collected N Comments FeLV 100-150 ug GoatSerum, 26.7 L 15 All goats achieved titer gp70 (6) milk* serum, over2000000^(#)  300 L milk Histophilus 150 ug Goat Serum 4.84 L 8 All goatsachieved titer somni (4) over 2500000. Two (H. somni) achieved titerover p31 protein 5000000 Bovine 61.8 ug Goat Serum 3.19 L 4 All goatsachieved titer Parainfluenza-3 (4) over 2000. Three (BPI-3) HN goatsachieved titer protein over 8000 rBVD1 150 ug Goat Serum 3.93 L 3 Allgoats achieved titer E2 (gp53) (4) over 100000. One goat consistentlyhad titers over 1200000 Canine 150 ug Goat Serum 4.51 L 4 All goatsachieved titer Circovirus (4) over 500000. Two antigen achieved titerover 2000000 Bordetella FHA 100 ug Goat Serum 2.57 L 4 All goatsachieved titer protein (4) 800,000. Two achieved titer over over 40000Parapoxvirus 150 ug Goat Serum 1.35 L 6 All goats achieved titer(inactivated) (4) over 4000000 *One goat in the group developed pseudopregnancy and lactated. 300 L of milk was collected from this goat.^(#)Titers deduced from the ELISA assays run. At that time endpointtiters were not indicated and the serum was not diluted out far enoughto determine endpoint.

Serum antibodies for FeLV gp70 were successfully purified using eitherProtein A or Protein G columns in-house, for small scale purification,or Protein G chromatography for large scale purification, at MaineBiotechnology Services (MBS), Portland, Me. The polyclonal antibodieswere concentrated using Millipore 30K Ultra Filter Units to a finalconcentration of about 1 mg/ml. Antibodies against the other antigensdisclosed in table 46 were unpurified.

The antibodies were isolated from milk obtained from a spontaneouslylactating goat immunized with FeLVgp70 according to a method comprisingthe following steps:

a) The pH of the milk was titrated to 4.6 with 2 M HCl and stirred atroom temp for 30 minutes for casein precipitation;

b) The milk was centrifuged at 17,000×g for 30 minutes and thesupernatant was collected;

c) Equilibration buffer was added to the supernatant to 3.3 M NaCl, 0.3M glycine and 0.2 M Tris base;

d) The supernatant was clarified by centrifugation at 3000×g for 15minutes;

e) The clarified milk supernatant was applied to a MabSelect columnequilibrated with the buffer in step ‘c’;

f) The column was washed with Equilibration buffer and eluted with 0.15Mglycine pH3.0;

g) Elute fractions were neutralized with 0.2 M Na phosphate.

As non-limiting examples, methods of generating anti-PI-3 and anti FeLVgp70 are provided below.

One of the objectives was to generate goat polyclonal antisera topurified Bovine Parainfluenza-3 (BPI-3) HN protein for use in in-vitroassays. This study was designed to vaccinate goats with a purifiedBovine Parainfluenza-3 (BPI-3) HN protein formulated with TXO adjuvantBovine Parainfluenza-3 (BPI-3) HN protein used as the antigen.

At approximately seven weeks after the first injection, it wasdetermined that all four of the goats had high enough serum antibodyconcentrations (SN over 1000) to begin production bleeds for serum.Production bleeds began one week after the fourth immunization. Bloodwas collected for serum at weekly intervals for three weeks. Serum fromeach goat was pooled for individual production bleeds. A total of3,187.50 mL of serum was collected during approximately 3 weeks ofproduction bleeds. Serum was processed and stored at −80° C. forevaluation in BPI-3 HN-based assays.

All serum collected from three goats (#30, 31 and 35) were thawed atroom temperature. Serum from goat number 34 was not used due to the lowantibody response in the screening ELISA. See Table 47 (PI3-NHPolyclonal antibody production: SN Response and antigen potency ELISA).

Goat number 35, collected 20 November 13, had the lowest volume ofavailable serum, 117 mL. Thus, 117 mL from each goat per collection waspooled in a sterile 1 L Nalgene PETG bottle. Approximately 1053 mL(9×117 mL) of serum was dispensed into 20, 50 mL aliquots in sterile 60mL Nalgene PETG bottles and 50, 1.0 mL aliquots.

TABLE 47 Animal ID with PI3 SN titer Immunization 30 31 34 35 0 <2 76 <2<2 1 215 1218 54 362 2 4096 8192 2435 9192 3 8192 16384 2696 9742

As a result, this study at completion successfully generated a total of3,187.5 mL of whole blood harvested from four goats that were repeatedlyimmunized with a purified BPI-3 protein formulated with TXO, during athree week period of production bleeds. Good polyclonal antibody titerswere generated in serum. Sufficient quantities of purified reagent wereobtained to for use in in-vitro assay applications.

In 2010, USDA notified industry that the FeLV gp85/70 capture reagentused for LEUKOCELL® and VERSIFEL® assays would no longer be supplied.Thus, the objective of this study was to generate goat polyclonalantisera to recombinant FeLV gp70 protein for use in in-vitro assays.

Previous attempts to produce antibody following vaccination withFreund's adjuvant were not successful. This study was designed tovaccinate goats with a 444 amino acid fragment of recombinant E.coli-expressed FeLV gp70 protein formulated with adjuvant. Beginningwith the 4th injection, the injection dose was reduced to 100 μg FeLVgp70 protein (instead of the original dose of 282 μg FeLV gp70 protein).The dose change was made because initial dose at 282 μg/mL was causinghigh incidence of injection site reactions. Dose was initially loweredto 100 μg/mL, but then raised to 150 μg/mL for the seventh immunizationand remained at that level until the end of the study (total of 15immunizations). PBS buffer was used to make up the difference in dosevolume, which remained at 1 mL.

Blood was collected from the goats, and once antibody titers weredetermined by direct and sandwich ELISA assay to be sufficiently high,serum was harvested and polyclonal antibodies were purified.

Six healthy female goats of LaMancha and Alpine breeds that were 1-3years of age and weighed>100 lbs were obtained for use in this study.Goats were fed hay and grain and had ad libitum access to waterthroughout the study. General health observations were performed oncedaily. A 1 mL dose of the experimental vaccine was administeredsubcutaneously to each goat at 21-day intervals, with a total of 15immunizations administered to each of the five goats that completed thestudy. Immunizations were initially administered in the neck or rearleg, alternating sides and sites for subsequent immunizations. Smallvisible injection site reactions were observed following immunizations.The immunization, administered to goats in the loose skin just cranialof the right rear leg, was reported to cause a little swelling,tenderness, and moderate lameness in all goats the next day. Subsequentinjections were administered in alternate sides of the neck or the areaover the ribs and were generally well tolerated. However, the area overthe ribs was ultimately found to be the location best tolerated by thegoats.

At approximately eight weeks after the first injection it was determinedthat four of the six goats (21, 22, 24, 25) had high enough serumantibody concentrations to begin production bleeds for serum. Productionbleeds from the remaining two goats (23, 26) were initiated five weekslater. Blood was collected for serum at weekly intervals. Goat 25 wasremoved from the study after six weeks of production blood collections.She was lame at arrival and displayed persistent lameness despiteBanamine treatments. Euthanasia was specified for the terminal bleed andadministered per site procedure, to ensure maximum blood volumecollection. Serum from each goat was pooled for individual productionbleeds. A total of 26.7 L of serum was collected during approximately 7months of production bleeds.

Unexpectedly, Goat 24 developed a pseudo-pregnancy during the study.Milk was collected from this goat for >3 months, with a total of 300 Lof milk available for antibody harvest. A protocol was developed forhigh-level purification of FeLV gp70 polyclonal antibody from the milk.

Antibodies were purified on two different dates from 500 mL of pooledserum from Goat 24 using Protein G Affinity Chromatography at MaineBiotechnology Services. A total of 6388 mg (321 mL of 19.9 mg/mL) and7343 mg (348 ml of 21.1 mg/ml) of purified goat anti-FeLV gp70antibodies were prepared for evaluation in FeLV-based assays.

Blood samples (approximately 25 mL/sample) were collected into 12.5 mLserum separatortubes (SST) fourteen days after each vaccination todetermine antibody concentrations to FeLV gp70. SSTs were labeled withthe goat ID and date of collection. Once assays determined that the FeLVgp70 antibody titers based on ELISA signal intensity for an animal wasat an acceptable concentration, production collections began from thatanimal. The blood volumes extracted from each goat were determined onthe basis of the goat's weight to obtain the maximum blood volume. IACUCguidelines allow for the collection of up to 7.5% of the blood volumeweekly.

Blood was collected into 12.5 mL SST for production collections. At theconclusion of the study, goats were euthanized by terminalexsanguinations and that blood was also collected for antibodyharvesting. All tubes were labeled with the goat's ID and the date ofcollection.

Blood was allowed to clot at room temperature. Following centrifugation,serum was harvested and transferred to polypropylene vials. Serum fromdifferent SST collected from the same goat on a collection day waspooled. Serum was held on ice until shipped for purification. A summaryof production is provided in table 48.

TABLE 48 Weekly Terminal Total Goat ID Volumes (ml) Bleed (ml)production (ml) 21  75-125 1000 4430 22  85-150 875 4665 23  85-125 10003630 24 110-200 1580 6785 25  80-105 980 1565 26 125-200 1750 6125 Total26.7 L

Goat 24 was producing serum with the highest antibody concentrations ofall the goats.

Purified serum antibodies from Goat 24 compared to the USDA 94-06 as acapture reagent using the FeLV gp70 C11D8 detection mAb in a SandwichELISA assay showed a similar dose response. The purified serum from Goat24 was compared to the USDA 94-06 reagent by Western Blot for theability to detect the FeLV gp85/70 protein. A similar Western Blotprofile was observed between the current capture, 94-06 and the new Goat24 capture, except that an additional ^(˜)15 kD band was observed withthe Goat 24 capture. Data using the new capture antibody showed that thecurrent reference has a different dose response curve shape than thecurrent reagent when used to capture the reference and the serialtested.

Both anrendati-FeLV gp70 purified from serum and milk functioned well asa capture reagent in the FeLV ELISA assays.

Additional studies are under way, as provided in Table 49.

It is expected that each of the formulations (antigens disclosed inTable 49 and adjuvanted with TXO) would elicit sufficiently highserology titers (over 1000, or more preferably, over 5000, or morepreferably, over 10000, or more preferably, over 50000, or morepreferably, over 100000, or more preferably, over 250000, or morepreferably, over 500000, or more preferably, over 1000000) in at leastone animal (preferably at least 2 animals, or more preferably in atleast three animals, or most preferably, in every animal treated) thusresulting in sufficient amount of antibodies for diagnostic or researchapplications.

TABLE 49 Amount Source Animal Total Serum Immunizations, Antigen(ug)/dose (N) Source of ABs Volume Collected N Clostridium 100 ug Goat(3) Serum 3.31 L/ongoing 10 perfringens Alpha toxin (inactivated)Clostridium 100 ug Goat (3) Serum 3.93 L/ongoing 9 perfringens Betatoxin (inactivated) Clostridium 100 ug Goat (3) Serum 4.07 L/ongoing 10perfringens Epsilon toxin (inactivated) 150 ug Goat (4) Serum 6/ongoingrBVD2 E2 (gp53) purified protein Brachyspira 150 ug Goat (3) Serum3/ongoing hyodysenteriae (strain B204) whole cell inactivatedBrachyspira 150 ug Goat (3) Serum 3/ongoing hyodysenteriae (BR2019-12strain) Pepsin digest

All publications cited in the specification, both patent publicationsand non-patent publications, are indicative of the level of skill ofthose skilled in the art to which this invention pertains. All thesepublications are herein fully incorporated by reference to the sameextent as if each individual publication were specifically andindividually indicated as being incorporated by reference.

Although the invention herein has been described with reference toparticular embodiments, it is to be understood that these embodimentsare merely illustrative of the principles and applications of thepresent invention. It is therefore to be understood that numerousmodifications may be made to the illustrative embodiments and that otherarrangements may be devised without departing from the spirit and scopeof the present invention as defined by the following claims.

1-34. (canceled)
 35. A vaccine composition comprising a Rhipicephalusmicroplus antigen and an adjuvant, said adjuvant being selected from thegroup consisting of: a) an aqueous adjuvant comprising animmunostimulatory oligonucleotide, a saponin, a sterol, a quaternaryamine, a polyacrylic polymer, and a glycolipid; and b) an oil-basedadjuvant, comprising an oily phase present in the amount of at least 45%v/v of the vaccine composition and comprising an immunostimulatoryoligonucleotide and a polycationic carrier, wherein said vaccinecomposition is a water-in-oil emulsion.
 36. The vaccine composition ofclaim 35, wherein the saponin is Quil A, the sterol is cholesterol, thequaternary amine is DDA, the glycolipid isN-(2-Deoxy-2-L-leucylamino-b-D-glucopyranosyl)-N-octadecyldodecanoylamideor a salt thereof, and the immunostimulatory oligonucleotide is a CpG.37. The vaccine composition of claim 35, wherein the polycationiccarrier is dextran DEAE and the immunostimulatory oligonucleotide is aCpG.
 38. A method treatment or prevention of an infection caused byRhipicephalus microplus, said method comprising administering to asubject in need thereof a vaccine composition according to claim 35.39-44. (canceled)
 45. The vaccine composition of claim 35 wherein theoily phase comprises at least 48% v/v of said vaccine composition. 46.The vaccine composition of claim 35 wherein the oily phase comprises atleast 50% v/v of said vaccine composition.
 47. the vaccine compositionof claim 46 wherein the oily phase comprises 50-52% of said vaccinecomposition.